CN112237134A - Rapid breeding method of bacterial wilt-resistant mulberry variety - Google Patents
Rapid breeding method of bacterial wilt-resistant mulberry variety Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G2/00—Vegetative propagation
- A01G2/10—Vegetative propagation by means of cuttings
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
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Abstract
The invention belongs to the technical field of plant variety breeding, and particularly relates to a rapid breeding method of a bacterial wilt-resistant mulberry variety. The method comprises the following steps: selecting branches of the fruit mulberry, carrying out nursery stock culture, carrying out water culture by using a bacterial liquid of ralstonia solanacearum after 80-100 days of culture, carrying out water culture for 30-45 days, and finally obtaining the fruit mulberry variety with the bacterial wilt resistance, carrying out planting culture on the fruit mulberry variety with the bacterial wilt resistance in a bacterial wilt serious disease area, wherein the planting age is 2-3 years, and then the fruit mulberry variety with the bacterial wilt resistance can be bred, wherein the bacterial liquid of ralstonia solanacearum is prepared by using a strain G12-9 or a strain G12-50. Compared with the traditional method for culturing bacterial wilt-resistant mulberry varieties, the method greatly reduces the waste amount of mulberry materials, remarkably reduces the workload of variety breeding, and overcomes the problems of long breeding period, complex operation, difficult identification and the like of the bacterial wilt-resistant mulberry varieties in the prior art.
Description
Technical Field
The invention belongs to the technical field of plant variety breeding, and particularly relates to a rapid breeding method of a bacterial wilt-resistant mulberry variety.
Background
Bacterial wilt is a devastating disease caused by ralstonia solanacearum, which is widely distributed in tropical, subtropical and some warm areas of the world. The host range of the ralstonia solanacearum is very wide, hundreds of plants of 44 families can be infected by the ralstonia solanacearum, and new hosts are continuously discovered, so that great economic loss is brought to crop production.
At present, one of effective methods for preventing the diffusion of ralstonia solanacearum is to adopt sterile plant propagation materials, and also to detect the ralstonia solanacearum in plants, soil and water samples through the rapid detection of the ralstonia solanacearum, so as to discover and eliminate the pathogenic bacteria in time and prevent the spreading of the pathogenic bacteria.
The mulberry bacterial wilt is a vascular bundle disease which causes leaf dehydration and mulberry death due to the fact that bacterial wilt infects xylem ducts of mulberry roots to affect water transportation, is fiercely and quickly spread, is a destructive disease of soil-borne plants of the mulberry and can cause death of new mulberry or grown-up mulberry in the same year.
However, as a new health-care fruit, fresh mulberry, mulberry juice, mulberry wine, mulberry dried fruit and other processed products are more and more favored by consumers, in recent years, the development of the mulberry industry in China is fast, the growing area of the mulberry is rapidly increased, and in order to protect the variety of the mulberry from being damaged by bacterial wilt, research and development of the variety of the mulberry are dedicated to reduce the probability of being damaged by the bacterial wilt.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a rapid breeding method of a bacterial wilt-resistant variety of fruit mulberry, which realizes disease control of the bacterial wilt of the fruit mulberry variety by breeding the fruit mulberry variety with bacterial wilt resistance.
The technical content of the invention is as follows:
the invention provides a rapid breeding method of a bacterial wilt-resistant mulberry variety, which comprises the following steps: selecting branches of fruit mulberry, carrying out nursery stock culture, carrying out water culture by using bacterial liquid of ralstonia solanacearum after 80-100 days of culture, carrying out water culture for 30-45 days, selecting the finally surviving fruit mulberry variety with the bacterial wilt resistance, carrying out planting culture on the disease-serious area with the bacterial wilt resistance by using the fruit mulberry variety with the bacterial wilt resistance, wherein the planting age is 2-3 years, and then breeding the fruit mulberry variety with the bacterial wilt resistance;
the water culture comprises early stage water culture, middle stage water culture and later stage water culture, and the water culture time of the three stages is 10-15 days;
the ralstonia solanacearum bacterial liquid is prepared by adopting a strain G12-9 (the strain preservation number KJ 494377) or a strain G12-50 (the strain preservation number KJ 494378);
the bacterial liquid concentration of the ralstonia solanacearum used in early stage water culture is 0.1 multiplied by 109cfu/mL~0.5×109cfu/mL, the ambient temperature is 30-35 ℃, and the ambient humidity is 70-85%;
the bacterial liquid concentration of the ralstonia solanacearum used for the middle-stage water culture and the later-stage water culture is 0.8 multiplied by 109cfu/mL~1×109cfu/mL, the ambient temperature is 30-35 ℃, and the ambient humidity is 70-85%;
the operation of water culture is that the roots of the mulberry seedlings are obliquely cut and then are directly soaked in the bacterial liquid of ralstonia solanacearum, so that the contact area of the roots of the mulberry seedlings is increased;
the preparation method of the ralstonia solanacearum bacterial liquid comprises the following steps:
1) selecting mulberry diseased plants infected by bacterial wilt: cutting mulberry root, peeling and taking xylem;
2) and (3) pathogenic bacteria separation: soaking mulberry root in alcohol, then washing with sterile water, then soaking in NaClO, then washing with sterile water, and placing into an EP tube filled with sterile water to obtain a primary bacterial liquid, and taking the primary bacterial liquid to perform culture medium coating culture to obtain primary bacteria;
3) pathogen purification:
4) and amplifying and storing pathogenic bacteria.
And 3) the original germ purification comprises the step of coating or streaking the bacterial colony obtained in the step 2) on a TTC solid medium flat plate again for purification, and culturing in a climatic chamber.
The invention has the following beneficial effects:
compared with the traditional method for culturing the bacterial wilt-resistant mulberry variety, the rapid breeding method for the bacterial wilt-resistant mulberry variety greatly reduces the waste amount of the mulberry material, remarkably reduces the workload of variety breeding, obviously shortens the time required for breeding the disease-resistant mulberry by the method, only needs 2-3 years, needs 6-8 years for the traditional breeding method, and overcomes the problems of long breeding period, complex operation, difficult identification and the like of the existing bacterial wilt-resistant mulberry variety;
the rapid breeding method is simple and easy to operate and remarkable in effect, the adopted staged hydroponics method is beneficial to accelerating the screening of disease-resistant varieties, the screening is accurate, the method can be effectively applied to rapid breeding of bacterial wilt-resistant mulberry varieties, and the obtained varieties are excellent in quality.
Detailed Description
The present invention is described in further detail in the following detailed description with reference to specific embodiments, which are intended to be illustrative only and not to be limiting of the scope of the invention, as various equivalent modifications of the invention will become apparent to those skilled in the art after reading the present invention and are intended to be included within the scope of the appended claims.
All the raw materials and reagents of the invention are conventional market raw materials and reagents unless otherwise specified.
Example 1
A rapid breeding method of a bacterial wilt-resistant mulberry variety comprises the following steps:
1) selecting Guangdong fruit mulberry resource as breeding material, selecting variety resource (all existing in south China garden) including fruit 04-76, fruit 04-78, fruit 04-25, Yuguo 06-15, fruit 04-7, fruit 04-74, Yuguo 06-7, fruit 04-111, fruit 04-34, fruit 04-43, Yuguo 04-35, fruit 04-52, fruit 04-75, fruit 04-62, Yuguo 06-5, fruit 04-73, fruit 04-97, fruit 04-46, fruit 04-93, fruit 04-52, fruit 04-72, fruit 04-132, fruit 04-139, fruit 04-136, fruit 04-137, fruit 04-117, fruit 04-75, fruit, 04-125 parts of fruit selection, 04-129 parts of fruit selection, 04-120 parts of fruit selection, 04-123 parts of fruit selection, 04-122 parts of fruit selection, 06-11 parts of fruit selection, 06-10 parts of Yu fruit, 06-16 parts of Yu fruit, 06-8 parts of Yu fruit, 04-12 parts of fruit selection, 06-11 parts of Yu fruit, 04-14 parts of fruit selection, 06-13 parts of Yu fruit, 04-19 parts of fruit selection, 04-20 parts of fruit selection, 04-88 parts of fruit selection, 04-106 parts of fruit selection, 04-15 parts of fruit selection, 04-102 parts of fruit selection, 04-105 parts of fruit selection, 04-87 parts of fruit selection, 04-61 parts of fruit selection, 04-49 parts of fruit selection, 04-8 parts of fruit selection, 06-17 parts of Yu fruit selection, 04-60 parts of fruit selection, 04-30 parts of fruit selection, 04-70 parts of fruit selection, 04-71 parts of fruit selection, 04-40 parts of fruit selection, 04-37 parts of fruit selection, fruit selection 04-58, fruit selection 04-77, fruit selection 04-94, fruit selection 04-107, fruit selection 04-28, fruit selection 04-55, fruit selection 04-33, fruit selection 04-26, fruit selection 04-51 and yuguo 06-19;
2) cultivating nursery stock with branch of the fruit mulberry variety by cuttage, cutting off the root of the nursery stock obtained after 80 days of cultivation, directly soaking in bacterial liquid of ralstonia solanacearum for water cultivation, wherein the early stage water cultivation adopts 0.2 × 10 concentration9carrying out water culture on cfu/mL G12-50 ralstonia solanacearum bacterial liquid for 10 days at the temperature of 30-32 ℃ and the environmental humidity of 70-77%;
the medium-term water culture adopts the concentration of 0.8 multiplied by 109carrying out water culture on cfu/mL G12-50 ralstonia solanacearum bacterial liquid for 10 days at the temperature of 30-33 ℃ and the ambient humidity of 75-81%;
the later stage water culture adopts the concentration of 1.0 multiplied by 109carrying out water culture on cfu/mL G12-50 ralstonia solanacearum bacterial liquid for 15 days at the temperature of 33-35 ℃ and the ambient humidity of 77-85%;
3) the fruit mulberry variety which is screened out by water culture and has the bacterial wilt resistance comprises 04-15 parts of fruit selection, 04-19 parts of fruit selection, 04-55 parts of fruit selection, 04-49 parts of fruit selection, 04-77 parts of fruit selection, 04-87 parts of fruit selection, 04-107 parts of fruit selection, 04-123 parts of fruit selection, 04-132 parts of fruit selection, 04-106 parts of fruit selection, 04-102 parts of fruit selection and 06-16 parts of Yuguo;
4) and (3) planting and culturing the mulberry variety with the bacterial wilt resistance in a bacterial wilt resistant disease-seriously diseased area, and performing disease resistance field identification, wherein the planting age is 2 years, and finally, breeding 5 mulberry varieties with bacterial wilt resistance, high yield and high quality, namely, 04-19 fruit varieties, 04-55 fruit varieties, 04-49 fruit varieties, 04-87 fruit varieties and 04-123 fruit varieties.
Example 2
A rapid breeding method of a bacterial wilt-resistant mulberry variety comprises the following steps:
1) same as example 1, step 1);
2) cultivating nursery stock with branch of the fruit mulberry variety by cuttage, cutting the nursery stock obtained after 90 days of cultivation, cutting the root of the nursery stock obliquely, directly soaking in bacterial liquid of ralstonia solanacearum for water cultivation, wherein the early stage water cultivation adopts 0.4 × 10 concentration9carrying out water culture on cfu/mL G12-9 ralstonia solanacearum bacterial liquid for 10 days at the temperature of 30-32 ℃ and the environmental humidity of 70-75%;
the concentration of the medium-term water culture is 0.9 multiplied by 109carrying out water culture on cfu/mL G12-9 ralstonia solanacearum bacterial liquid for 10 days at the temperature of 32-34 ℃ and the environmental humidity of 76-80%;
the later stage water culture adopts the concentration of 1.0 multiplied by 109carrying out water culture on cfu/mL G12-9 ralstonia solanacearum bacterial liquid for 10 days at the temperature of 33-35 ℃ and the environmental humidity of 78-85%;
3) the fruit mulberry variety which is screened out by water culture and has the bacterial wilt resistance comprises 04-15 parts of fruit selection, 04-19 parts of fruit selection, 04-55 parts of fruit selection, 04-49 parts of fruit selection, 04-77 parts of fruit selection, 04-87 parts of fruit selection, 04-107 parts of fruit selection, 04-123 parts of fruit selection, 04-132 parts of fruit selection, 04-106 parts of fruit selection, 04-102 parts of fruit selection and 06-16 parts of Yuguo;
4) and (3) planting and culturing the variety of the fruit mulberry with the bacterial wilt resistance in a bacterial wilt heavy land, performing disease resistance field identification, wherein the planting age is 3 years, and finally breeding 5 varieties of the fruit mulberry with the bacterial wilt resistance, high yield and high quality, namely, 04-19 fruit choices, 04-55 fruit choices, 04-49 fruit choices, 04-87 fruit choices and 04-123 fruit choices.
Example 3
A rapid breeding method of a bacterial wilt-resistant mulberry variety comprises the following steps:
1) same as example 1, step 1);
2) cutting the branches of the mulberry variety by a cuttage methodCultivating nursery stock, beveling the nursery stock root after 100 days of cultivation, directly soaking in bacterial liquid of ralstonia solanacearum for water cultivation, wherein the early water cultivation adopts 0.5 × 10 concentration9carrying out water culture on cfu/mL G12-50 ralstonia solanacearum bacterial liquid for 13 days at the temperature of 30-32 ℃ and the environmental humidity of 70-75%;
the concentration of the medium-term water culture is 1.0 multiplied by 109carrying out water culture on cfu/mL G12-50 ralstonia solanacearum bacterial liquid for 14 days at the temperature of 31-33 ℃ and the environmental humidity of 74-79%;
the later stage water culture adopts the concentration of 0.9 multiplied by 109carrying out water culture on cfu/mL G12-50 ralstonia solanacearum bacterial liquid for 13 days at the temperature of 32-35 ℃ and the environmental humidity of 78-85%;
3) the fruit mulberry variety which is screened out by water culture and has the bacterial wilt resistance comprises 04-15 parts of fruit selection, 04-19 parts of fruit selection, 04-55 parts of fruit selection, 04-49 parts of fruit selection, 04-77 parts of fruit selection, 04-87 parts of fruit selection, 04-107 parts of fruit selection, 04-123 parts of fruit selection, 04-132 parts of fruit selection, 04-106 parts of fruit selection, 04-102 parts of fruit selection and 06-16 parts of Yuguo;
4) and (3) planting and culturing the fruit mulberry variety with the bacterial wilt resistance in a bacterial wilt heavy land, performing disease resistance field identification, wherein the planting age is 2.5 years, and finally breeding 5 fruit mulberry varieties with bacterial wilt resistance, high yield and high quality, wherein the fruit selection is 04-19, the fruit selection is 04-55, the fruit selection is 04-49, the fruit selection is 04-87 and the fruit selection is 04-123.
Example 4
A rapid breeding method of a bacterial wilt-resistant mulberry variety comprises the following steps:
1) same as example 1, step 1);
2) cultivating nursery stock with branch of the fruit mulberry variety by cuttage, cutting off the root of the nursery stock obtained after 95 days of cultivation, directly soaking in bacterial liquid of ralstonia solanacearum for water cultivation, wherein the early stage water cultivation adopts 0.3 × 10 concentration9carrying out water culture on cfu/mL G12-9 ralstonia solanacearum bacterial liquid for 15 days at the temperature of 30-33 ℃ and the environmental humidity of 70-75%;
the medium-term water culture adopts the concentration of 0.8 multiplied by 109carrying out water culture on cfu/mL G12-9 ralstonia solanacearum bacterial liquid for 15 days at the temperature of 32-34 ℃ and the ambient humidity of 76-81%;
the later stage water culture adopts the concentration of 0.9 multiplied by 109Water culture of cfu/mL G12-9 ralstonia solanacearum bacterial liquid for 15 daysThe temperature is 33-35 ℃, and the environmental humidity is 80-85%;
3) the fruit mulberry variety which is screened out by water culture and has the bacterial wilt resistance comprises 04-15 parts of fruit selection, 04-19 parts of fruit selection, 04-55 parts of fruit selection, 04-49 parts of fruit selection, 04-77 parts of fruit selection, 04-87 parts of fruit selection, 04-107 parts of fruit selection, 04-123 parts of fruit selection, 04-132 parts of fruit selection, 04-106 parts of fruit selection, 04-102 parts of fruit selection and 06-16 parts of Yuguo;
4) and (3) planting and culturing the fruit mulberry variety with the bacterial wilt resistance in a bacterial wilt heavy land, performing disease resistance field identification, wherein the planting age is 2.5 years, and finally breeding 5 fruit mulberry varieties with bacterial wilt resistance, high yield and high quality, wherein the fruit selection is 04-19, the fruit selection is 04-55, the fruit selection is 04-49, the fruit selection is 04-87 and the fruit selection is 04-123.
Comparative example 1
By taking the example 1 as a comparison, the water culture in the step 2) is changed into the prior art, the early stage is 100 days of nursery seedling culture, the later stage is 35 days of water culture, and the concentration of G12-50 ralstonia solanacearum bacterial liquid is 1.0 multiplied by 109cfu/mL, other steps are unchanged;
the fruit mulberry varieties with bacterial wilt resistance screened by water culture comprise 04-15 fruit selection, 04-19 fruit selection, 04-77 fruit selection, 04-87 fruit selection, 04-123 fruit selection, 04-132 fruit selection, 04-106 fruit selection and 04-102 fruit selection;
4) and (3) planting and culturing the fruit mulberry variety with the bacterial wilt resistance in a bacterial wilt heavy land, performing disease resistance field identification, wherein the planting age is 6 years, and finally breeding 2 fruit mulberry varieties with bacterial wilt resistance, high yield and high quality, namely, 04-19 fruit varieties and 04-123 fruit varieties.
Comparative example 2
By contrast, in example 1), the concentration of G12-50 bacterial liquid of ralstonia solanacearum used for hydroponics in step 2) was 1.0X 109Water culture is carried out for 35 days in cfu/mL, and other steps are unchanged;
selecting fruit mulberry varieties with bacterial wilt resistance by water culture, wherein the fruit mulberry varieties comprise 04-15 parts of fruit selection, 04-19 parts of fruit selection, 04-55 parts of fruit selection, 04-77 parts of fruit selection, 04-87 parts of fruit selection, 04-107 parts of fruit selection, 04-123 parts of fruit selection and 04-132 parts of fruit selection;
4) and (3) planting and culturing the fruit mulberry variety with the bacterial wilt resistance in a bacterial wilt heavy land, performing disease resistance field identification, wherein the planting age is 3.5 years, and finally breeding 3 high-yield and high-quality fruit mulberry varieties, namely, 04-19 fruit varieties, 04-55 fruit varieties and 04-123 fruit varieties.
Therefore, the same variety of the fruit mulberry is adopted in the above embodiments, and the finally bred fruit mulberry varieties with bacterial wilt resistance, high yield and high quality by the adopted method are the same varieties, which shows that the breeding method of the invention is effective, has pertinence, and can efficiently breed the fruit mulberry variety with bacterial wilt resistance.
The preparation of the ralstonia solanacearum bacterial liquid in the embodiment comprises the following steps:
(1) pretreatment of samples
Samples taken from the mulberry field are stored by using a sample bag, and the sampling time, the sampling place, the morbidity degree of the mulberry and the sampling part are recorded. The samples should be stored in an ice box in the field, and separated as soon as possible in the same day, otherwise, the samples should be stored in a refrigerator at 4 ℃ and separated within 3 days. Cleaning the retrieved mulberry root or stem (hereinafter root is taken as a representative), and cutting off a part of mulberry root for later use;
(2) isolation of pathogenic bacteria
The operation process is carried out on a clean bench, and the relevant tools and vessels are preferably used after high-temperature and high-pressure sterilization.
Firstly, 0.3-1.0cm of the cleaned mulberry root is cut, and the xylem is taken out after the mulberry root is peeled;
② immersing in 75 percent alcohol for about 10 s, taking out and washing with sterile water for 3 times, immersing in 10 percent NaClO solution for about 30 s, taking out and washing with sterile water for 3 times, and putting into an EP tube (sterilized) filled with 1ml of sterile water. Soaking for 15-30 min, and oscillating for 1-2 min to obtain original bacterial liquid;
③ taking out 100 mul of original bacteria liquid by a liquid transfer device, adding the original bacteria liquid into an EP tube filled with 900 mul of sterile water for dilution, and respectively carrying out the method according to 10 times on the original bacteria-1、10-2、10-3、10-4、10-5Diluting in a gradient manner for plate coating;
fourthly, 10 is taken-3、10-4、10-5Respectively coating 100 mul of the diluted bacterial liquid on prepared TTC solid culture medium plates;
finally, the coated plate is placed in a climatic chamber with the temperature of 30 ℃ and the humidity of 70 percent for culturing for 48 to 72 hours and observed. If no bacterial colony or too little bacterial colony exists on the flat plate, the concentration of the coated bacterial liquid can be amplified, even the bacterial liquid can be coated by original bacterial liquid, the bacterial liquid prepared at the early stage can be stored in a refrigerator at 4 ℃ for standby, preferably not more than 48h, because the bacteria die gradually in the process of placement;
(3) purification of pathogenic bacteria
Generally, after 48h of culture, obvious colonies can be basically appeared, and parts of the colonies grow slowly and need 72h of culture. At this time, the colonies can be preliminarily observed and preliminarily classified according to the colony morphology. Different colonies were re-plated or streaked on TTC solid medium plates for purification, and cultured in a climatic chamber under the same conditions as in the isolation step. The colony cultured again is generally a single colony, can be used for observing the colony morphology, and can also be amplified by an LB liquid culture medium for strain preservation;
(4) amplification of pathogenic bacteria
In a clean bench, taking purified bacterial colony to prepare bacterial liquid, or directly picking bacterial colony, inoculating in 200 or 250ml triangular flask or conical flask, wherein 100ml LB liquid culture medium can be, but is not more than half of volume, preventing liquid from splashing, and sealing with multilayer gauze. Placing the inoculated conical flask in a temperature-controlled shaking table, culturing for 48h at the temperature of 30 ℃ and at the temperature of 170-;
(5) preservation of pathogenic bacteria
Taking purified and identified bacterial colonies, and carrying out amplification culture by using an LB liquid culture medium, such as amplification of pathogenic bacteria;
mixing 750 mu lLB culture medium bacterial liquid with 250 mu l sterilized pure glycerin by using a sterilized EP tube and an adjustable micropipette, and fully mixing by using an oscillator;
marking the strain number by a marking pen, freezing and storing the strain according to the principle of slow freezing and fast melting, and finally storing the strain in an ultra-low temperature refrigerator at minus 80 ℃;
and fourthly, when the preserved bacteria are taken out and activated, distilled water with the temperature of about 40 ℃ is used, so that the preserved strains are quickly thawed, the death of a large amount of bacteria is avoided, and the bacteria can be naturally thawed but not beneficial to being preserved again.
Claims (8)
1. A rapid breeding method of a bacterial wilt-resistant mulberry variety is characterized by comprising the following steps: selecting branches of fruit mulberry, carrying out nursery stock culture, carrying out water culture by using bacterial liquid of ralstonia solanacearum after 80-100 days of culture, carrying out water culture for 30-45 days, selecting the fruit mulberry variety which finally survives and has the bacterial wilt resistance, carrying out planting culture on the disease-serious area of the bacterial wilt by using the fruit mulberry variety with the bacterial wilt resistance, wherein the planting age is 2-3 years, and then breeding the fruit mulberry variety with the bacterial wilt resistance.
2. The rapid breeding method of the bacterial wilt resistant mulberry variety of claim 1, wherein the water culture comprises early stage water culture, middle stage water culture and later stage water culture, and the water culture time of the three stages is 10-15 days.
3. The method for rapidly breeding the bacterial wilt-resistant mulberry variety as claimed in claim 1, wherein the bacterial liquid of ralstonia solanacearum is prepared from strain G12-9 or strain G12-50.
4. The method for rapidly breeding the bacterial wilt-resistant mulberry variety as claimed in claim 2, wherein the bacterial liquid concentration of the ralstonia solanacearum used in earlier stage water culture is 0.1X 109cfu/mL~0.5×109cfu/mL, the ambient temperature is 30-35 ℃, and the ambient humidity is 70-85%.
5. The method of producing a bacterial wilt-resistant mulberry variety of claim 2The rapid breeding method is characterized in that the concentration of the ralstonia solanacearum bacterial liquid used for middle-stage water culture and later-stage water culture is 0.8 multiplied by 109cfu/mL~1×109cfu/mL, the ambient temperature is 30-35 ℃, and the ambient humidity is 70-85%.
6. The rapid breeding method of the bacterial wilt resistant mulberry variety of claim 1, wherein the water culture operation is that the root of the mulberry seedling is obliquely cut and then directly soaked in the bacterial liquid of ralstonia solanacearum.
7. The method for rapidly breeding the bacterial wilt-resistant mulberry variety of claim 1, wherein the preparation of the bacterial liquid of ralstonia solanacearum comprises the following steps:
1) selecting mulberry diseased plants infected by bacterial wilt: cutting mulberry root, peeling and taking xylem;
2) and (3) pathogenic bacteria separation: soaking mulberry root in alcohol, then washing with sterile water, then soaking in NaClO, then washing with sterile water, and placing into an EP tube filled with sterile water to obtain a primary bacterial liquid, and taking the primary bacterial liquid to perform culture medium coating culture to obtain primary bacteria;
3) pathogen purification:
4) and amplifying and storing pathogenic bacteria.
8. The method for rapidly breeding the bacterial wilt-resistant mulberry variety as claimed in claim 7, wherein the purification of the original pathogen in step 3) comprises the steps of coating or streaking the colony obtained in step 2) again on a TTC solid medium plate for purification, and culturing in a climatic chamber.
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| CN115976151A (en) * | 2022-12-12 | 2023-04-18 | 中国农业科学院油料作物研究所 | Water culture inoculation method for rapidly and efficiently identifying peanut bacterial wilt resistance |
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| CN102517376A (en) * | 2011-12-13 | 2012-06-27 | 广东省农业科学院蚕业与农产品加工研究所 | Method for identifying resistance to bacterial wilt disease of mulberry |
| CN103194524A (en) * | 2013-04-03 | 2013-07-10 | 山东省花生研究所 | Method for rapidly identifying peanut germplasm with bacterial wilt resistance |
| CN107211861A (en) * | 2017-07-04 | 2017-09-29 | 四川省农业科学院经济作物育种栽培研究所 | The method of seedling stage Rapid identification tobacco bred Resistance to bacterial wilt |
| CN108476981A (en) * | 2018-03-27 | 2018-09-04 | 中国热带农业科学院热带生物技术研究所 | A kind of screening technique of anti-blight banana germplasm |
| CN110169327A (en) * | 2019-06-10 | 2019-08-27 | 广西壮族自治区农业科学院经济作物研究所 | The method for building up in bacterial wilt of peanut Resistance Identification garden |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115976151A (en) * | 2022-12-12 | 2023-04-18 | 中国农业科学院油料作物研究所 | Water culture inoculation method for rapidly and efficiently identifying peanut bacterial wilt resistance |
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