Some standard content:
National Standard of the People's Republic of China
Rice Seed Origin Quarantine Procedure
Plant quarantine tules for producing areas of rice seeds This procedure applies to the origin quarantine of rice seed breeding bases. 1 Glossary
UDC 633. 183-152
GB 8371—87
1.1 Origin quarantine: refers to the quarantine during the rice seed production process. Including the selection of bases, the selection of disease-free varieties, inspections during the growth period, necessary indoor inspections, etc., until the issuance of the "Origin Quarantine Certificate". 1.2 Quarantine objects: refers to dangerous pests and diseases that are prohibited from being transmitted by inspection seeds, seedlings, plant residues, etc. according to national laws. 2 Quarantine objects
2. 1 Xanthomonas Campestris pv. Oryzicola (Fang et al) Dye.2.2 Xanthomonas Campestris pv. Oryzae (ISHIYAMA) Dye.3 Selection of bases
Never occurred or did not occur for three consecutive years, with certain isolation and protection conditions, and irrigation water sources are not contaminated by quarantine objects. 4 Requirements for seeds used in bases
4.1 Seeds used in bases should be selected from areas where no quarantine objects occur or seeds that have been certified by the quarantine department as not containing quarantine objects. 4.2 Seeds must be disinfected before sowing (see Appendix A for details). 5 Comprehensive management measures
5.1 Requirements for cultivation management
5.1.1 The fields should be selected in high and dry places upstream of the irrigation system and far away from the village. 5.1.2 The straw used to bundle the seedlings should be sterilized in boiling water for 30 minutes, or it can be replaced by wheat straw, palm leaves, etc. 5.1.3 Use pesticides for prevention and control 1~~~~2 times in the three-leaf stage and 3~~~5 days before transplanting. 5.1.4 Irrigation
5.1.4.1 Separate irrigation and drainage. bZxz.net
5.1.4.2 Irrigate frequently with shallow water, strictly prevent cross-irrigation, deep water irrigation, and immersion irrigation, and timely irrigate the field. 5.1.5 Fertilization
5.1.5.1 The base fertilizer is fully decomposed.
5.1.5.2 Prevent the partial application of nitrogen fertilizer, and the nitrogen, phosphorus and potassium should be reasonably proportioned to prevent the rice from being greedy for green and inducing diseases. 5.2 Treatment of diseased fields
5.2.1 Mark the diseased fields and the diseased centers. 5.2.2 Chemical control: Once the disease center is found in the field, immediately pull out or cut off the diseased plants, spray pesticides to control, and spray pesticides for prevention in the surrounding fields, especially after storms and floods. 5.3 Treatment of rice seeds in diseased fields
Approved by the Ministry of Agriculture of the People's Republic of China on November 25, 1987 and implemented on July 1, 1988
GB8371-87
5.3.1 Seeds produced in diseased fields should be collected and stored separately to prevent mixing with disease-free seeds. 5.3.1.1 Seeds from lightly diseased fields (only sporadic diseased leaves) must be disinfected and strictly controlled for use in diseased areas (see 5.2 for seed disinfection methods).
5.3.1.2 Seeds from other diseased fields should be converted into food and not used as seeds. 5.4 Treatment of diseased rice straw: Diseased rice straw should be burned as fuel or sterilized in other ways. Do not use diseased straw to tie seedlings and do not apply disease-stopping fertilizers to rice fields.
6 Inspection and testing methods
6.1 Field inspection
6.1.1 Field inspection time
6.1.1.1 Seedling fields should be visually inspected from the four-leaf stage. If suspicious disease spots are found, the diseased seedlings should be pulled out for identification. 6.1.1.2 The field should be inspected three times: the first time is at the jointing stage; the second time is from the booting stage to the heading stage, which is usually the epidemic period of the disease, with obvious symptoms and easy to identify; the third time is from the time when the heading is complete to the time when the leaves turn yellow, combined with seed purity and quality inspection. 6.1.2 Field inspection methods
6.1.2.1 Inspection method: Check the seed field piece by piece and strip by strip (including the isolation area). 6.1.2.2 Symptom diagnosis: Identification of field symptoms (see Appendix B for details). For suspicious symptoms, collect specimens and bring them back to the room for identification. 6.2 Indoor inspection method
6.2.1 Bacteria overflow inspection: Cut about 1 mm of leaf tissue at the junction of diseased and healthy leaves on the diseased leaves, place it in the water droplets on the slide, add a cover glass, let it stand for 1 to 2 minutes, and then use a handheld magnifying glass or a low-power microscope to check under a slightly dark field of view. There will be cloudy bacterial liquid flowing out from the leaf veins. 6.2.2 Moisture inspection: Take a glass cup, put clean river sand about half an inch deep in it, add water to moisten it, and then cut about 6.6 cm of diseased leaf tissue, insert the lower end into the river sand, expose the upper end, cover with wet gauze, and moisturize for more than 12 hours. Turbid liquid or light yellow beads will form at the upper incision. 6.2.3 Staining inspection: Cut off the stem of the diseased leaf, insert it into a glass tube or wide-mouth bottle filled with red water (original solution or diluted), and place it in a ventilated and warm place. After 30 minutes, the healthy part will be dyed red, and the diseased part will remain green or yellow because the duct is full of bacteria, which affects the entry of red ink. 6.2.4 Isolation and culture inspection: Isolation and culture of diseased tissue for bacteriological inspection (see Appendix C for details). 6.3 Inspection of seed with bacteria: Use phage method or immunofluorescence method to inspect the seed with bacteria (see Appendix D for details). 7 Issuance of certificate
7.1 After the last field inspection, if no rice seeds of quarantine objects are found, the local quarantine department shall issue a "Origin Quarantine Certificate" (see Appendix E).
7.2 Seeds produced in seed fields with quarantine objects will not be issued with "Origin Quarantine Certificate". 7.3 Seed departments purchase disease-free seeds with "Origin Quarantine Certificate". 150
A1 Bacterial leaf streak disease, white leaf blight.
GB8371—87
Appendix A
Disinfection treatment method for rice seeds
(reference)
A1.1 Hot water blanching: Put the rice seeds in a basket, put them in 45℃ warm water to preheat for 3 minutes, and immediately move them into 58℃ hot water to soak for 10 minutes. After taking out the rice, immediately rinse it with cold water to cool it, then soak the seeds for germination or dry them for sowing. A1.280% "402" 1000 times (or 10% "401\500 times) + 0.4% hydrochloric acid (chemically pure) for 36 hours. (Note: According to the results of field application, the soaking time is 36 h is relatively safe. In actual application, different regions should conduct germination tests on different varieties before application). A1.3 Chloramphenicol 500ppm is planted for 48h (water temperature 25~28℃). Appendix B
Field symptom identification
(Supplement)
B1 Rice bacterial leaf streak
Rice bacterial leaf streak forms dark green or yellow-brown narrow stripes on the leaves. In the initial stage, it is dark green water-like translucent small spots, which quickly stretch between the leaf veins to form stripes about 1/3~3/4mm wide and 1~4mm long. It can be expanded to 1mm wide and more than 10mm long, turning yellow-brown. The lesions are overflowed with strings of yellow bead-like bacteria, which are small in shape but large in quantity. In severe cases, the number of lesions increases and fuses together, and the local area appears irregular yellow-brown to white spots. When observed against the light, the lesions are translucent and water-like. The diseased part has a lot of bacterial glue, dark color, and is not easy to fall off. Fall. Typical symptoms can be seen in the seedling stage.
B2 Rice Bacterial Leaf Blight
B2.1 Typical Symptoms of Bacterial Leaf Blight
Typical symptoms of bacterial leaf blight mainly occur in leaves and leaf sheaths. After the pathogen invades from the tip or edge of the leaf, it first produces yellow-green, water-soaked stripes and spots, and then continues to develop into wavy yellow, yellow-green or gray-green lesions along one or both sides of the leaf edge or the midrib of the leaf. The boundary between diseased and healthy tissues is obvious. The lesions turn grayish white after a few days and curl inwards, and look dry from a distance. When the air visibility is high or the dew point is high in the evening and early morning after rain, turbid water droplets or dense yellow glue-like bacterial pus will be spit out on the fresh lesions on the diseased leaves, and sometimes even on the leaf edges that do not show lesions. After drying, they harden into granules and are easy to fall off. In some susceptible varieties, the lesions can spread from the leaves downward to the leaf sheaths, such as yellow-white turning to grayish white, and the leaf sheaths will eventually dry up. B2.2 Symptoms of wilt-type bacterial leaf blight
Symptoms of wilt-type bacterial leaf blight mainly occur in the seedling field and before and after transplantation. After the young plants are infected, they can gradually show symptoms in the late growth period of the seedlings or within 1 to 4 weeks after transplantation. The symptoms are mainly caused by the invasion of pathogens from leaf wounds, natural orifices, stem injuries or broken roots, and then transfer to other organs along the vascular bundle in the field, secrete toxins to destroy and block the transport tissues, causing water loss in the seedling field and wilt of the whole plant. In the early stage of the occurrence of wilt, water-soaked gray-green acute lesions first appear on the invasion point of the pathogen (water hole or wound on the tip of the leaf), and then quickly extend downward along the main vein to the leaf sheath. The infected leaves and leaf sheaths quickly dry up, and even die and fall off. After 7 to 10 days, wilt plants will appear, the heart leaves will curl, and the shape will be like a dead heart. However, as long as the withered plants are pulled up, the withered heart leaves are peeled off, and the browning part of the pseudostem of the diseased plants is dissected, and the broken ends are squeezed by hand, a large amount of yellow-white to yellow snot-like bacterial pus can be seen overflowing. When the inner side of the leaf sheath of the diseased plants is peeled off, a large amount of bacterial pus can also be seen overflowing. If the pathogen invades from the stem or broken roots, and the sheath wounds penetrated by the new roots extending from the root point, it can quickly and directly invade the heart leaves or pseudostem tissues. The wilt appears early, and the stem leaves or heart leaves can curl one after another due to the serious development of symptoms; but sometimes leaf blight-type lesions often appear at the wounds on the elongated heart leaves. The symptoms of wilt and typical white leaf blight can be transformed into each other, occurring alone or at the same time. 151
B2.3 Yellow leaf symptoms
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Yellow leaf symptoms are often seen on the new leaves at the heart of mature plants, which turn pale yellow to greenish yellow after infection, while other lower leaves are normal green. It is generally difficult to detect bacteria on yellow leaf diseased leaves; however, the internodes and pseudostems below the infected leaves are heavily infected, and when the conducting tissues of the stem nodes are not yet damaged, they can still supply a small amount of nutrients to the young leaves, causing the leaves to be half-dead and pale yellow. Appendix C
Bacteriological test methods
(Supplement)
The isolation method of rice bacterial leaf blight and rice leaf streak is the same as that of general plant pathogenic bacteria, except that sucrose is used instead of glucose in the culture medium. The cultured blood after separation is first stored in a 20-25℃ incubator for one day and then transferred to a 27-29℃ incubator for culture. The appearance of colonies generally takes 3-7 days. In terms of the physiological and biochemical reactions of bacteria, leaf streak and rice bacterial leaf blight are basically similar, but there are several significant differences: (1) Gelatin liquefaction: rice bacterial leaf blight cannot liquefy, while leaf streak can liquefy gelatin. (2) Cow milk culture: rice bacterial leaf blight cannot age, while leaf streak can age. (3) Arabinose fermentation: rice bacterial leaf blight cannot utilize it and does not produce acid, while leaf streak can utilize it and produce acid. (4) Xanthoceras spp. are sensitive to reducing substances and cannot grow on a medium containing 2% glucose, while septoria spp. are insensitive to reducing substances and can grow on a medium containing 2% glucose. (5) The serum and phage reactions of the two are different. Appendix D
Testing methods for infected seeds
(Supplementary)
D1 Phage testing method
D1.1 Testing time: The tested rice seeds should be tested within three months after harvest. D1.2 Sample preparation: Sample rice seeds at multiple points, and after thorough mixing, randomly weigh 10g of seeds, remove the husks, grind them, and put them into a sterilized beaker or mortar, add 20mL of sterilized water, soak and stir frequently, and after half an hour, draw the supernatant for determination. It can also be filtered with coarse filter paper to remove tissue residues, and the filtrate is taken for determination.
D1.3 Preparation of indicator bacteria solution: Use OS-3 (Jiangsu) and OS-14 (Liaoning) strains as mixed indicator bacteria (local strains can also be used when testing local seed materials). To ensure the purity of the indicator bacteria, the indicator bacteria should be freshly cultured, especially those cultured on slant surfaces. Generally, bacteria transplanted on slant surfaces for 3 to 5 days are the best. Bacteria that have grown for more than 10 days should not be used. The concentration of indicator bacteria suspension should not be too dilute, and it is better to have a bacterial count of more than 900 million per milliliter. Add 5 mL of sterile water to each slant bacterial tube, scrape off the bacterial moss, and prepare a bacterial suspension for determination.
D1.4 Determination and counting: For each sample, take 1.0, 1.0, and 0.5 mL of the upper solution and place them in three sterile culture plates, add 1 mL of indicator bacteria solution and 10 mL of dissolved solid culture medium, shake well to form a plate, and place it in a 25~~28℃ incubator. After culturing for 10~12 hours, record the number of plaques in each culture plate, and then convert it into the number of plaques per gram of seeds. D2 Fluorescent antibody test method
D2.1 Fluorescent antibody test for rice bacterial blight species Operating procedures D2.1.1 Preparation of fluorescent film
D2.1.1.1 Weigh 5g of dry rice seeds (preferably within one year after harvest), hull them and set aside. D2.1.1.2 Place the rice husk in a large test tube, add 10~15mL of physiological saline, and stir it evenly with a glass rod until it covers the rice husk, and incubate it in a 28℃ incubator for 2 hours.
GB 8371-87
D2.1.1.3 Stir the rice husk water and filter out the husks. Put the rice husk water into a centrifuge tube and centrifuge it at 1000r/min for 5min. Discard the sediment and keep the supernatant.
D2. 1. 1. 4
Put the supernatant into a centrifuge tube and centrifuge it at 4000r/min for 30min. Use a pipette to discard the supernatant and keep 0.5mL of the enriched bacterial solution at the bottom.
D2.1.1.5 Smear the enriched bacterial solution on the slide, dry it in the shade and quickly pass it over the flame of an alcohol lamp 1 to 2 times to fix the bacteria on the slide. D2.1.1.6 Cover the bacterial smear area on the slide with 1:10~15 antiserum against white leaf blight, place the slide horizontally in a sealed water-soluble box at 37℃ and keep it moist for 30min.
D2.1.1.7 Take out the slide from the previous item, rinse out the excess antiserum with phosphate buffer, and then dry it in the shade or blow it dry with a hair dryer. D2.1.1.8 Add 1:15 goat anti-rabbit fluorescent antibody to the bacterial smear area of the previous item slide, cover the smear area, and place it horizontally in a 37℃ water-soluble box for 30 minutes as before.
D2.1.1.9 Take out the slide, rinse the fluorescent antibody with phosphate buffer, dry it in the shade or blow it dry with a hair dryer, and set it aside. D2.1.2 Inspection of fluorescent slides (performed in the dark) D2.1.2.1 Add a drop of pH 8 buffered glycerol (or non-fluorescent microscope oil) to the bacterial smear area of the fluorescent slide. D2.1.2.2 Use the 100× objective lens of the fluorescence microscope to focus on the bacterial smear area of the slide, and carefully make the lens touch the glass slide. D2.1.2.3 Observe the eyepiece and use the fine adjustment screw to slowly move the objective lens up. When the eyepiece is brighter, pay attention to the yellow-green bright bacteria on the dark-field slide. Select 10 points to count the bacterial bodies of white leaf blight (short rods, thicker and fatter) (the relationship between the amount of bacteria in seeds and the incidence in the field needs to be further studied). Note: Use 0.8% sterilized saline to dilute antiserum and fluorescent antibodies. D2.2 Reagent formula
D2.2, 1 pH8.0 buffered glycerol (a substitute for non-fluorescent microscope oil) 5 parts of glycerol (first-level specification) + 1 part of pH8.0 concentration 0.01M phosphate buffer, mix well, and store in the refrigerator. D2.2.2 0.01M pH8.0 phosphate buffer formula stock solution: 0.2M phosphate buffer formula is as follows: 15.6g sodium dihydrogen phosphate (containing 2 crystal waters) is dissolved in 500mL water. 35.82g disodium hydrogen phosphate (containing 12 crystal waters) is dissolved in 500mL water. Take 5.3 mL of D2.2.2.1.
94.7 mL of D2.2.2.2.
17 g of sodium chloride is added with water to 2000 mL to form a 0.1 M, pH 8.0 phosphate buffer. If necessary, use 0.5 M sodium carbonate to correct the pH value. Additional remarks:
This standard was drafted by the National Plant Protection Station, Shaanxi Provincial Plant Protection Station, and Guangdong Provincial Plant Protection Station. The main drafters of this standard are Geng Bingjin, Wang Chunlin, Zhang Peihe, Zeng Dongsheng, and Yin Kaifeng. 1533 Yellow leaf symptoms
GB 8371 - 87
Yellow leaf symptoms are more common in the new leaves at the heart of mature plants, which turn pale yellow to greenish yellow after infection; while other lower leaves turn normal green. It is generally difficult to detect bacteria on yellow leaf diseased leaves; however, the internodes and pseudostems below the infected leaves carry a large number of bacteria, which can still supply a small amount of nutrients to the young leaves before the conductive tissue of the stem nodes is damaged, causing the leaves to turn into a semi-dead pale yellow state. Appendix C
Bacteriological test methods
(Supplementary)
The isolation method of rice bacterial blight and leaf streak bacteria is the same as that of general plant pathogenic bacteria, except that sucrose is used instead of glucose in the culture medium. The cultured blood after isolation is first stored in a 20-25℃ incubator for one day and then transferred to a 27-29℃ incubator for culture. The appearance of colonies generally takes 3-7 days. In terms of the physiological and biochemical reactions of bacteria, leaf streak pathogens and bacterial leaf blight pathogens are basically similar, but there are several significant differences: (1) Gelatin liquefaction: bacterial leaf blight pathogens cannot liquefy, while leaf streak pathogens can liquefy gelatin. (2) Cultivation of milk: bacterial leaf blight pathogens cannot age, while leaf streak pathogens can age. (3) Arabinose fermentation: bacterial leaf blight pathogens cannot utilize it and do not produce acid, while leaf streak pathogens can utilize it and produce acid. (4) Bacterial leaf blight pathogens are sensitive to reducing substances and cannot grow on a medium containing 2% glucose, while leaf streak pathogens are insensitive to reducing substances and can grow on a medium containing 2% glucose. (5) The serum reactions and phage reactions of the two pathogens are different. Appendix D
Testing methods for infected seeds
(Supplement)
D1 Phage testing method
D1.1 Testing time: The tested rice seeds should be tested within three months after harvest. D1.2 Sample preparation: Sample rice seeds at multiple points, and after thorough mixing, randomly weigh 10g of seeds, remove the husks, grind and put into a sterilized beaker or mortar, add 20mL of sterilized water, soak and stir frequently, and absorb the upper clear liquid for determination after half an hour. It can also be filtered with coarse filter paper to remove tissue residues, and the filtrate is taken for determination.
D1.3 Preparation of indicator bacteria liquid: Use OS-3 (Jiangsu) and OS-14 (Liaoning) strains as mixed indicator bacteria (local strains can also be used when testing local seed materials). To ensure the purity of indicator bacteria, indicator bacteria should be freshly cultured, especially those cultured on slant surfaces. Generally, bacteria transplanted on slant surfaces for 3 to 5 days are the best, and those over 10 days should not be used. The concentration of indicator bacteria suspension should not be too dilute, and it is better to have a bacterial count of more than 900 million per milliliter. Add 5mL of sterilized water to each slant bacterial tube, scrape off the bacterial moss, and prepare a bacterial suspension for determination.
D1.4 Determination and counting: For each sample, take 1.0, 1.0, and 0.5 mL of the upper solution and place them in three sterile culture plates, add 1 mL of indicator bacteria solution and 10 mL of dissolved solid culture medium, shake well to form a plate, and place it in a 25~~28℃ incubator. After culturing for 10~12 hours, record the number of plaques in each culture plate, and then convert it into the number of plaques per gram of seeds. D2 Fluorescent antibody test method
D2.1 Fluorescent antibody test for rice bacterial blight species Operating procedures D2.1.1 Preparation of fluorescent film
D2.1.1.1 Weigh 5g of dry rice seeds (preferably within one year after harvest), hull them and set aside. D2.1.1.2 Place the rice husk in a large test tube, add 10~15mL of physiological saline, and stir it evenly with a glass rod until it covers the rice husk, and incubate it in a 28℃ incubator for 2 hours.
GB 8371-87
D2.1.1.3 Stir the rice husk water and filter out the husks. Put the rice husk water into a centrifuge tube and centrifuge it at 1000r/min for 5min. Discard the sediment and keep the supernatant.
D2. 1. 1. 4
Put the supernatant into a centrifuge tube and centrifuge it at 4000r/min for 30min. Use a pipette to discard the supernatant and keep 0.5mL of the enriched bacterial solution at the bottom.
D2.1.1.5 Smear the enriched bacterial solution on the slide, dry it in the shade and quickly pass it over the flame of an alcohol lamp 1 to 2 times to fix the bacteria on the slide. D2.1.1.6 Cover the bacterial smear area on the slide with 1:10~15 antiserum against white leaf blight, place the slide horizontally in a sealed water-soluble box at 37℃ and keep it moist for 30min.
D2.1.1.7 Take out the slide from the previous item, rinse out the excess antiserum with phosphate buffer, and then dry it in the shade or blow it dry with a hair dryer. D2.1.1.8 Add 1:15 goat anti-rabbit fluorescent antibody to the bacterial smear area of the previous item slide, cover the smear area, and place it horizontally in a 37℃ water-soluble box for 30 minutes as before.
D2.1.1.9 Take out the slide, rinse the fluorescent antibody with phosphate buffer, dry it in the shade or blow it dry with a hair dryer, and set it aside. D2.1.2 Inspection of fluorescent slides (performed in the dark) D2.1.2.1 Add a drop of pH 8 buffered glycerol (or non-fluorescent microscope oil) to the bacterial smear area of the fluorescent slide. D2.1.2.2 Use the 100× objective lens of the fluorescence microscope to focus on the bacterial smear area of the slide, and carefully make the lens touch the glass slide. D2.1.2.3 Observe the eyepiece and use the fine adjustment screw to slowly move the objective lens up. When the eyepiece is brighter, pay attention to the yellow-green bright bacteria on the dark-field slide. Select 10 points to count the bacterial bodies of white leaf blight (short rods, thicker and fatter) (the relationship between the amount of bacteria in seeds and the incidence in the field needs to be further studied). Note: Use 0.8% sterilized saline to dilute antiserum and fluorescent antibodies. D2.2 Reagent formula
D2.2, 1 pH8.0 buffered glycerol (a substitute for non-fluorescent microscope oil) 5 parts of glycerol (first-level specification) + 1 part of pH8.0 concentration 0.01M phosphate buffer, mix well, and store in the refrigerator. D2.2.2 0.01M pH8.0 phosphate buffer formula stock solution: 0.2M phosphate buffer formula is as follows: 15.6g sodium dihydrogen phosphate (containing 2 crystal waters) is dissolved in 500mL water. 35.82g disodium hydrogen phosphate (containing 12 crystal waters) is dissolved in 500mL water. Take 5.3 mL of D2.2.2.1.
94.7 mL of D2.2.2.2.
17 g of sodium chloride is added with water to 2000 mL to form a 0.1 M, pH 8.0 phosphate buffer. If necessary, use 0.5 M sodium carbonate to correct the pH value. Additional remarks:
This standard was drafted by the National Plant Protection Station, Shaanxi Provincial Plant Protection Station, and Guangdong Provincial Plant Protection Station. The main drafters of this standard are Geng Bingjin, Wang Chunlin, Zhang Peihe, Zeng Dongsheng, and Yin Kaifeng. 1533 Yellow leaf symptoms
GB 8371 - 87
Yellow leaf symptoms are more common in the new leaves at the heart of mature plants, which turn pale yellow to greenish yellow after infection; while other lower leaves turn normal green. It is generally difficult to detect bacteria on yellow leaf diseased leaves; however, the internodes and pseudostems below the infected leaves carry a large number of bacteria, which can still supply a small amount of nutrients to the young leaves before the conductive tissue of the stem nodes is damaged, causing the leaves to turn into a semi-dead pale yellow state. Appendix C
Bacteriological test methods
(Supplementary)
The isolation method of rice bacterial blight and leaf streak bacteria is the same as that of general plant pathogenic bacteria, except that sucrose is used instead of glucose in the culture medium. The cultured blood after isolation is first stored in a 20-25℃ incubator for one day and then transferred to a 27-29℃ incubator for culture. The appearance of colonies generally takes 3-7 days. In terms of the physiological and biochemical reactions of bacteria, leaf streak pathogens and bacterial leaf blight pathogens are basically similar, but there are several significant differences: (1) Gelatin liquefaction: bacterial leaf blight pathogens cannot liquefy, while leaf streak pathogens can liquefy gelatin. (2) Cultivation of milk: bacterial leaf blight pathogens cannot age, while leaf streak pathogens can age. (3) Arabinose fermentation: bacterial leaf blight pathogens cannot utilize it and do not produce acid, while leaf streak pathogens can utilize it and produce acid. (4) Bacterial leaf blight pathogens are sensitive to reducing substances and cannot grow on a medium containing 2% glucose, while leaf streak pathogens are insensitive to reducing substances and can grow on a medium containing 2% glucose. (5) The serum reactions and phage reactions of the two pathogens are different. Appendix D
Testing methods for infected seeds
(Supplement)
D1 Phage testing method
D1.1 Testing time: The tested rice seeds should be tested within three months after harvest. D1.2 Sample preparation: Sample rice seeds at multiple points, and after thorough mixing, randomly weigh 10g of seeds, remove the husks, grind and put into a sterilized beaker or mortar, add 20mL of sterilized water, soak and stir frequently, and absorb the upper clear liquid for determination after half an hour. It can also be filtered with coarse filter paper to remove tissue residues, and the filtrate is taken for determination.
D1.3 Preparation of indicator bacteria liquid: Use OS-3 (Jiangsu) and OS-14 (Liaoning) strains as mixed indicator bacteria (local strains can also be used when testing local seed materials). To ensure the purity of indicator bacteria, indicator bacteria should be freshly cultured, especially those cultured on slant surfaces. Generally, bacteria transplanted on slant surfaces for 3 to 5 days are the best, and those over 10 days should not be used. The concentration of indicator bacteria suspension should not be too dilute, and it is better to have a bacterial count of more than 900 million per milliliter. Add 5mL of sterilized water to each slant bacterial tube, scrape off the bacterial moss, and prepare a bacterial suspension for determination.
D1.4 Determination and counting: For each sample, take 1.0, 1.0, and 0.5 mL of the upper solution and place them in three sterile culture plates, add 1 mL of indicator bacteria solution and 10 mL of dissolved solid culture medium, shake well to form a plate, and place it in a 25~~28℃ incubator. After culturing for 10~12 hours, record the number of plaques in each culture plate, and then convert it into the number of plaques per gram of seeds. D2 Fluorescent antibody test method
D2.1 Fluorescent antibody test for rice bacterial blight species Operating procedures D2.1.1 Preparation of fluorescent film
D2.1.1.1 Weigh 5g of dry rice seeds (preferably within one year after harvest), hull them and set aside. D2.1.1.2 Place the rice husk in a large test tube, add 10~15mL of physiological saline, and stir it evenly with a glass rod until it covers the rice husk, and incubate it in a 28℃ incubator for 2 hours.
GB 8371-87
D2.1.1.3 Stir the rice husk water and filter out the husks. Put the rice husk water into a centrifuge tube and centrifuge it at 1000r/min for 5min. Discard the sediment and keep the supernatant.
D2. 1. 1. 4
Put the supernatant into a centrifuge tube and centrifuge it at 4000r/min for 30min. Use a pipette to discard the supernatant and keep 0.5mL of the enriched bacterial solution at the bottom.
D2.1.1.5 Smear the enriched bacterial solution on the slide, dry it in the shade and quickly pass it over the flame of an alcohol lamp 1 to 2 times to fix the bacteria on the slide. D2.1.1.6 Cover the bacterial smear area on the slide with 1:10~15 antiserum against white leaf blight, place the slide horizontally in a sealed water-soluble box at 37℃ and keep it moist for 30min.
D2.1.1.7 Take out the slide from the previous item, rinse out the excess antiserum with phosphate buffer, and then dry it in the shade or blow it dry with a hair dryer. D2.1.1.8 Add 1:15 goat anti-rabbit fluorescent antibody to the bacterial smear area of the previous item slide, cover the smear area, and place it horizontally in a 37℃ water-soluble box for 30 minutes as before.
D2.1.1.9 Take out the slide, rinse the fluorescent antibody with phosphate buffer, dry it in the shade or blow it dry with a hair dryer, and set it aside. D2.1.2 Inspection of fluorescent slides (performed in the dark) D2.1.2.1 Add a drop of pH 8 buffered glycerol (or non-fluorescent microscope oil) to the bacterial smear area of the fluorescent slide. D2.1.2.2 Use the 100× objective lens of the fluorescence microscope to focus on the bacterial smear area of the slide, and carefully make the lens touch the glass slide. D2.1.2.3 Observe the eyepiece and use the fine adjustment screw to slowly move the objective lens up. When the eyepiece is brighter, pay attention to the yellow-green bright bacteria on the dark-field slide. Select 10 points to count the bacterial bodies of white leaf blight (short rods, thicker and fatter) (the relationship between the amount of bacteria in seeds and the incidence in the field needs to be further studied). Note: Use 0.8% sterilized saline to dilute antiserum and fluorescent antibodies. D2.2 Reagent formula
D2.2, 1 pH8.0 buffered glycerol (a substitute for non-fluorescent microscope oil) 5 parts of glycerol (first-level specification) + 1 part of pH8.0 concentration 0.01M phosphate buffer, mix well, and store in the refrigerator. D2.2.2 0.01M pH8.0 phosphate buffer formula stock solution: 0.2M phosphate buffer formula is as follows: 15.6g sodium dihydrogen phosphate (containing 2 crystal waters) is dissolved in 500mL water. 35.82g disodium hydrogen phosphate (containing 12 crystal waters) is dissolved in 500mL water. Take 5.3 mL of D2.2.2.1.
94.7 mL of D2.2.2.2.
17 g of sodium chloride is added with water to 2000 mL to form a 0.1 M, pH 8.0 phosphate buffer. If necessary, use 0.5 M sodium carbonate to correct the pH value. Additional remarks:
This standard was drafted by the National Plant Protection Station, Shaanxi Provincial Plant Protection Station, and Guangdong Provincial Plant Protection Station. The main drafters of this standard are Geng Bingjin, Wang Chunlin, Zhang Peihe, Zeng Dongsheng, and Yin Kaifeng. 1531 Weigh 5g of dry rice seeds (preferably within one year after harvest), hull them and set aside. D2.1.1.2 Place the rice husks in a large test tube, add 10-15mL of physiological saline, until the rice husks are covered, stir with a glass rod, and incubate in a 28℃ incubator for 2h.
GB 8371-87
D2.1.1.3 Stir the rice husk water, filter out the husks and discard them. Place the rice husk water in a centrifuge tube, centrifuge at 1000r/min for 5min, discard the sediment and keep the supernatant.
D2. 1. 1. 4
Put the supernatant in a centrifuge tube, centrifuge at 4000r/min for 30min, discard the supernatant with a pipette, and keep 0.5mL of the enriched bacterial liquid at the bottom.
D2.1.1.5 Apply the enriched bacterial solution to the slide, dry it in the shade, and quickly pass it over the flame of an alcohol lamp 1 to 2 times to fix the bacteria on the slide. D2.1.1.6 Cover the bacterial smear area on the slide with 1:10~15 antiserum of white leaf blight, place the slide horizontally in a 37℃ sealed water-soluble box, and keep it moist for 30 minutes.
D2.1.1.7 Take out the slide from the previous item, rinse out the excess antiserum with phosphate buffer, and then dry it in the shade or with a hair dryer. D2.1.1.8 Add 1:15 goat anti-rabbit fluorescent antibody to the bacterial smear area of the slide from the previous item, cover the smear area, and place it horizontally in a 37℃ water-soluble box for 30 minutes as before.
D2.1.1.9 Take out the slide, rinse the fluorescent antibody with phosphate buffer, dry it in the shade or with a hair dryer, and set it aside. D2.1.2 Fluorescence slide inspection (conducted in the dark) D2.1.2.1 Drop a drop of pH 8 buffered glycerol (or non-fluorescent microscope oil) on the bacterial area of the fluorescent slide. D2.1.2.2 Use the 100× objective lens of the fluorescence microscope to aim at the bacterial area of the slide, and carefully make the lens touch the glass slide. D2.1.2.3 Observe the eyepiece, use the fine adjustment screw to slowly move the objective lens up, and when the eyepiece is brighter, pay attention to observe the yellow-green bright bacteria on the dark-view slide, and select 10 points to count the bacterial bodies of white leaf blight (short rods, thicker and fatter) (the relationship between the amount of bacteria in seeds and the incidence of field diseases needs to be further studied). Note: 0.8% sterilized saline is used to dilute antiserum and fluorescent antibodies. D2.2 Reagent formula
D2.2, 1pH8.0 buffered glycerol (a substitute for non-fluorescent microscope oil) 5 parts of glycerol (first-level specification) + 1 part of pH8.0 concentration 0.01M phosphate buffer, mix well, and store in the refrigerator. D2.2.20.01MpH8.0 phosphate buffer solution formula: 0.2M phosphate buffer solution formula is as follows: 15.6g sodium dihydrogen phosphate (containing 2 crystal waters) dissolved in 500mL water. 35.82g disodium hydrogen phosphate (containing 12 crystal waters) dissolved in 500mL water. Take D2.2.2.15.3 mL.
D2.2.2.2 94.7 mL.
Add 17g sodium chloride to 2000mL to obtain 0.1M, pH8.0 phosphate buffer. If necessary, use 0.5M sodium carbonate to correct the pH value. Additional remarks:
This standard was drafted by the National Plant Protection Station, Shaanxi Provincial Plant Protection Station, and Guangdong Provincial Plant Protection Station. The main drafters of this standard are Geng Bingjin, Wang Chunlin, Zhang Peihe, Zeng Dongsheng, and Yin Kaifeng. 1531 Weigh 5g of dry rice seeds (preferably within one year after harvest), hull them and set aside. D2.1.1.2 Place the rice husks in a large test tube, add 10-15mL of physiological saline, until the rice husks are covered, stir with a glass rod, and incubate in a 28℃ incubator for 2h.
GB 8371-87
D2.1.1.3 Stir the rice husk water, filter out the husks and discard them. Place the rice husk water in a centrifuge tube, centrifuge at 1000r/min for 5min, discard the sediment and keep the supernatant.
D2. 1. 1. 4
Put the supernatant in a centrifuge tube, centrifuge at 4000r/min for 30min, discard the supernatant with a pipette, and keep 0.5mL of the enriched bacterial liquid at the bottom.
D2.1.1.5 Apply the enriched bacterial solution to the slide, dry it in the shade, and quickly pass it over the flame of an alcohol lamp 1 to 2 times to fix the bacteria on the slide. D2.1.1.6 Cover the bacterial smear area on the slide with 1:10~15 antiserum of white leaf blight, place the slide horizontally in a 37℃ sealed water-soluble box, and keep it moist for 30 minutes.
D2.1.1.7 Take out the slide from the previous item, rinse out the excess antiserum with phosphate buffer, and then dry it in the shade or with a hair dryer. D2.1.1.8 Add 1:15 goat anti-rabbit fluorescent antibody to the bacterial smear area of the slide from the previous item, cover the smear area, and place it horizontally in a 37℃ water-soluble box for 30 minutes as before.
D2.1.1.9 Take out the slide, rinse the fluorescent antibody with phosphate buffer, dry it in the shade or with a hair dryer, and set it aside. D2.1.2 Fluorescence slide inspection (conducted in the dark) D2.1.2.1 Drop a drop of pH 8 buffered glycerol (or non-fluorescent microscope oil) on the bacterial area of the fluorescent slide. D2.1.2.2 Use the 100× objective lens of the fluorescence microscope to aim at the bacterial area of the slide, and carefully make the lens touch the glass slide. D2.1.2.3 Observe the eyepiece, use the fine adjustment screw to slowly move the objective lens up, and when the eyepiece is brighter, pay attention to observe the yellow-green bright bacteria on the dark-view slide, and select 10 points to count the bacterial bodies of white leaf blight (short rods, thicker and fatter) (the relationship between the amount of bacteria in seeds and the incidence of field diseases needs to be further studied). Note: 0.8% sterilized saline is used to dilute antiserum and fluorescent antibodies. D2.2 Reagent formula
D2.2, 1pH8.0 buffered glycerol (a substitute for non-fluorescent microscope oil) 5 parts of glycerol (first-level specification) + 1 part of pH8.0 concentration 0.01M phosphate buffer, mix well, and store in the refrigerator. D2.2.20.01MpH8.0 phosphate buffer solution formula: 0.2M phosphate buffer solution formula is as follows: 15.6g sodium dihydrogen phosphate (containing 2 crystal waters) dissolved in 500mL water. 35.82g disodium hydrogen phosphate (containing 12 crystal waters) dissolved in 500mL water. Take D2.2.2.15.3 mL.
D2.2.2.2 94.7 mL.
Add 17g sodium chloride to 2000mL of water to make 0.1M, pH8.0 phosphate buffer. If necessary, use 0.5M sodium carbonate to correct the pH value. Additional remarks:
This standard was drafted by the National Plant Protection Station, Shaanxi Provincial Plant Protection Station, and Guangdong Provincial Plant Protection Station. The main drafters of this standard are Geng Bingjin, Wang Chunlin, Zhang Peihe, Zeng Dongsheng, and Yin Kaifeng. 153
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