CN108207612B - Efficient pear tree rot resistance breeding method - Google Patents
Efficient pear tree rot resistance breeding method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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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
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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Abstract
The invention belongs to the technical field of fruit tree breeding, and particularly relates to a high-efficiency pear tree rot resistance breeding method. The method comprises the following steps of selecting by hybridization combination, hybridizing and pollinating, layering hybrid seeds, sowing and transplanting, inoculating pear tree rotten germs, and eliminating susceptible plants according to the number of progeny plants in the hybridization combination: the number of single plants in the hybridization combination is more than 500, the single plants with the morbidity of more than 40 percent and the lesion length of more than 2cm are eliminated, the number of the single plants in the hybridization combination is less than 500, the single plants with the morbidity of more than 70 percent and the lesion length of more than 2cm are eliminated; and then selecting the plumest bud within 10cm of the topmost end of the resistant plant, and grafting the bud onto the birchleaf pear rootstock.
Description
Technical Field
The invention belongs to the technical field of fruit tree breeding, and particularly relates to a high-efficiency pear tree rot resistance breeding method.
Background
The pear tree rot disease (pear Valsa canker) is commonly called as 'rotten skin disease', is one of the most serious diseases in pear cultivation production, mainly damages a main trunk, a main branch and a side branch of a pear tree, causes bark rot, tree vigor weakness and reduces the yield and quality of pear fruits. The disease has the characteristics of wide occurrence area, high incidence rate and difficult control, and can cause serious pear orchard diseases, tree scab accumulation, incomplete branch and trunk defects, even a large number of dead trees or garden damage.
The pears are wide in the cultivation area of China, are one of the fruit tree species which are most widely cultivated in China, and the fruits are also one of the fruits which are deeply loved by consumers. Along with the development of the whole society and economy, people have higher and higher requirements on pomes, the appearance is required to be attractive, the quality of the pomes also needs to meet the requirements of different consumer groups, and variety breeding is the basis of variety improvement, so that more and more attention is paid. The fruit has beautiful appearance, excellent quality and strong resistance, and becomes the main breeding target of the pear. The pear breeding means comprises bud mutation seed selection, seedling seed selection, cross breeding, mutation breeding and the like, and at present, edible varieties cultivated through cross breeding in China account for 72% of new varieties, so the cross breeding is still the main method and approach for breeding new pear varieties.
The process of the hybrid breeding comprises the steps of making a breeding plan according to breeding targets, collecting pollen, pollinating, bagging and isolating, managing hybrid fruits, picking the hybrid fruits in autumn, picking seeds after the hybrid fruits are refrigerated, cleaning, drying and storing the hybrid seeds, performing hybrid seed lamination treatment, sowing, maintaining in seedling stage, planting or directly picking terminal buds for grafting, starting the hybrid seedlings to obtain the result after 5-7 years of field management, performing comprehensive evaluation on the fruits and the like, breeding excellent plants, checking, deciding and examining to form a new variety.
Because the pear trees are high in genetic heterozygosis, hybrid progeny has wide separation, and the pear trees can be obtained only by configuring a large hybrid population in order to obtain an ideal variety with multiple excellent traits. Meanwhile, the childhood period of the pear tree is long, the period from sowing of hybrid seeds to fruiting is usually 5-7 years, the field growth of 5-7 years is long, the period is long, the occupied area is large, the consumed manpower and material resources are large, the breeding period is shortened, the breeding efficiency is improved, and the pre-selection research on early-stage characters of fruit tree filial generations is always a focus of attention of breeders. If single plants which can become good varieties in the future can be selected in the seedling stage, the single plants without target characters or inferior characters are eliminated, and the breeding cost is greatly saved.
The resistance to the pear rot disease is greatly different among different varieties, which provides possibility for selecting the pear varieties resisting the rot disease through breeding. The use of resistant varieties to reduce the occurrence of rot is the most effective and safe measure in production to control rot. How to select the best strain from the hybrid seedlings as soon as possible by early selection in breeding is the key point of research work of many breeders.
In the prior art, the rot resistance is not selected in the early stage in the breeding process of the pear trees, so that the breeding process of the bred varieties is greatly limited, for example, the breeding process cannot be promoted in a rot disease area of the pear trees, and the resource is wasted; the Chinese invention patent CN201310468321.X discloses a method for identifying resistance of pear tree rot, but does not suggest how to combine resistance identification with early selection of breeding, and further research is needed for screening standards of resistant plants and the like.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a high-efficiency pear tree rot resistance breeding method.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a high-efficiency pear tree rot resistance breeding method, which comprises the following steps:
step 1: selecting a hybrid combination in which at least one parent has rot disease resistance to be more than resistant;
the resistance of the filial generation to rot disease is influenced by the parent variety, the resistance of the parent variety has great influence on the number of resistance plants of the filial generation, the number of disease-resistant plants in the filial generation of which parents are not the resistance variety is small, one of the parents or the parents is the disease-resistant variety, and more disease-resistant plants can be generated in the filial generation;
step 2: collecting buds of the male parent in a big balloon period, peeling off anthers, drying, keeping at the drying temperature of 22-25 ℃, sealing and storing at 4 ℃ after pollen scattering, pollinating the buds of the female parent in the big balloon period, immediately bagging and isolating, and removing the isolation after pollination for 10-14 days; bagging the fruits after 3 weeks of pollination, spraying the pesticide and the bactericide on the whole plant once in 1 week before bagging, and soaking the bag opening with 500 times of 70% thiophanate methyl solution and 1000 times of chlorpyrifos solution before bagging;
and step 3: harvesting the hybrid fruits which are physiologically mature, transporting the hybrid fruits back to the room to emit field heat, and then removing fruit bags; pre-cooling for 24 hours at the temperature of 15-20 ℃, then storing in a cold storage at the temperature of 0-2 ℃ for 60-80 days, taking out the hybrid seeds, scrubbing with warm water at the temperature of 25 ℃, then drying in the air, and storing at room temperature;
and 4, step 4: and (3) stacking hybrid seeds in the last 12 th or the last 1 st of the year: firstly, soaking hybrid seeds in warm water at 25 ℃ for 12 hours, then uniformly placing the seeds in a container with water-absorbing filter paper at the bottom, placing the container in a cold storage at 0-4 ℃, keeping the filter paper moist, and sowing the seeds in a special hole tray for forest trees with the depth of 11cm after 70-80% of the hybrid seeds are exposed to the white;
and 5: when 4 true leaves and root length of the hybrid seedling are 10cm, transplanting the hybrid seedling to a nursery land with the furrow width of 80cm, planting 4 rows in one furrow and planting in wide and narrow rows; cutting off branches and thorns on the trunk in 8 months of the transplanting current year, and re-cutting once in late 9 months;
step 6: and (3) inoculating pear tree canker in 8 last ten days of the year: firstly, culturing pear rot germs in an incubator at 25 ℃ for 4 days, then beating hyphae into fungus cakes by using a puncher, inoculating hybrid seedlings with the ground diameter of more than 1cm, and inoculating five fungus cakes every 15cm upwards from the ground height of 80cm of the hybrid seedlings during inoculation; the inoculation method comprises the steps of punching holes at the positions of 80cm, 95cm, 110cm, 125cm and 140cm high of the main stem of the hybrid seedling by using a puncher until the main stem is thick enough to touch the xylem, placing the back surface of a fungus cake on three layers of filter paper or absorbent cotton which is completely soaked by sterile water, enabling the surface with hyphae to completely correspond to the holes in the main stem of the hybrid seedling, and then completely winding the main stem of the hybrid seedling from bottom to top by using a preservative film; the pear tree rot disease bacteria culture medium is a PSA culture medium;
and 7: after 3 weeks of inoculation, the single plants with the scabs extending out of the preservative film are eliminated; after 4 weeks of inoculation, counting the disease incidence and the length of disease spots of each inoculation point, and calculating the disease incidence of each individual plant, wherein the disease incidence is the incidence of the inoculation point/the total inoculation point multiplied by 100%; the number of single plants in the hybrid combination is more than 500, and the single plants with the morbidity of more than 40 percent and the lesion length of more than 2cm are eliminated; the number of single plants in the hybrid combination is less than 500, and the single plants with the morbidity of more than 70 percent and the lesion length of more than 2cm are eliminated; shearing the eliminated hybrid seedlings from the roots, and then collecting and burning the hybrid seedlings in a centralized manner;
and 8: in spring of the next year, selecting the plumest bud within 10cm of the topmost end of the resistant single plant, and grafting the bud onto the pear stock with the planted plant-line spacing of 0.5m multiplied by 2 m; the topmost bud of the plant is firstly converted into reproductive growth, so that the grafted single plant can bloom and bear fruits within 2-3 years.
Further, the method also comprises the step of applying strong fruit fertilizer in 5 months of the current year of hybridization, wherein 30kg of urea, 30kg of potassium sulfate and 4000kg of biogas slurry water are applied to each mu.
Further, the drying temperature in the step 2 is 25 ℃; the segregation was removed after pollination for 14 d.
Further, in the step 2, the pesticide is 4000 times of 5% abamectin, and the bactericide is 800 times of 70% thiophanate methyl.
Further, in the step 3, the pre-cooling temperature is 15 ℃, the storage temperature is 0 ℃, and the storage time is 70 d.
Further, applying organic fertilizer for 4-7 m in a nursery garden before transplanting the hybrid seedlings in the step 53Urea 30 kg/mu; thoroughly watering the hybrid seedlings before transplanting, sprinkling irrigation and watering the seedlings after transplanting, topdressing from 5 middle ten days to 8 middle ten days of the year, fertilizing 1 time every 10 days, and applying 2-4 kg/mu of urea every time; and (3) removing weeds before topdressing, wherein the growth amount of the hybrid seedlings in the current year exceeds 1.5m, the height of the plants reaches 2.5-3 m, and the average ground diameter is larger than 1 cm.
Furthermore, each furrow in the step 5 is provided with 2 narrow lines and 1 wide line, the line spacing of the wide lines is 40cm, the line spacing of the narrow lines is 20cm, the planting distance is 15-20 cm, and the furrow spacing is 50 cm.
Compared with the prior art, the invention has the beneficial effects that:
1. compared with the existing breeding method, the method saves half of land, can bring the hybrid seedlings into the fruiting period 2-3 years ahead of time, and starts to evaluate in the later period.
2. The invention determines the standard of eliminated diseased plants when different cross combinations are large: when the number of single plants in the hybrid combination is less than 500, the single plants with the morbidity rate of more than 70% and the lesion length of more than 2cm are eliminated, and when the number of the single plants in the combination is more than 500, the single plants with the morbidity rate of more than 40% and the lesion length of more than 2cm are eliminated, so that a certain seedling amount of each combination can be ensured, the infected single plants can be eliminated in a nursery at the initial stage of breeding, and the land occupation amount and manpower and material resources in subsequent breeding are reduced.
3. In the invention, when the hybrid seedlings are transplanted to a nursery land, wide-narrow row planting is selected, the row spacing of the wide rows is 40cm, and the row spacing of the narrow rows is 20cm, so that the requirement of passing breeders during plant management in the breeding process can be met, the growth of pears can be met, the land is saved, and the cost is saved.
4. In the invention, after the hybrid seedlings are transplanted, branches and thorns on the trunk are cut off in 8 months in the current year, and the cutting is repeated once in the last ten days of 9 months, which brings great convenience to the passage and operation of breeders in the later hybrid breeding process and brings more convenience to the breeders.
5. The rot-resistant pear tree obtained by breeding by the method can be popularized in a disease area and is not limited by rot.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
A high-efficiency pear tree rot disease resistance breeding method comprises the following steps:
step 1: selecting a variety 'Xingao' with high susceptibility to rot as a female parent and a variety 'Xinyao' with rot resistance as a male parent;
step 2: collecting flower buds in big balloon period, stripping off anthers, collecting, drying at 25 deg.C, loosening pollen, sealing at 4 deg.C for temporary storage, pollinating at big balloon period of female parent flower buds, bagging for isolation immediately after pollination for 14d, and removing isolation; bagging the fruits after 3 weeks of pollination, and spraying a solution of 800 times of thiophanate methyl and a solution of 4000 times of abamectin 5% once on the whole plant within 1 week before bagging; and soaking the bag opening with 500 times of 70% thiophanate methyl solution and 1000 times of chlorpyrifos the day before bagging; when the bag is sleeved, the bag opening is firstly propped up, the bag bottom is propped up, the bag body is expanded, and after the fruit is sleeved, the bag opening is sealed around the fruit handle; applying fruit strengthening fertilizer 5 months in the year, and applying 30kg of urea, 30kg of potassium sulfate and 4000kg of biogas slurry water per mu;
and step 3: harvesting the hybrid fruits which are physiologically mature, transporting the hybrid fruits back to the room after picking to allow the hybrid fruits to emit field heat, and then removing fruit bags; precooling for 24h at 15 ℃, and then storing in a refrigeration house at 0 ℃; after 70 days of storage, taking the hybrid fruits out of a refrigeration house, transversely cutting the hybrid fruits, taking the hybrid seeds out of ventricles by using tweezers, collecting the hybrid seeds by using seed bags, then scrubbing the hybrid seeds at 25 ℃ in warm water, drying the seeds in the air after cleaning the seeds, and storing the seeds at room temperature;
and 4, step 4: the hybrid seeds are layered in 12 last ten days of the year: firstly, putting hybrid seeds in warm water at 25 ℃ for fully absorbing water for 12 hours, additionally preparing a preservation box, putting filter paper with full water absorption at the bottom of the box, uniformly and non-overlapping the hybrid seeds on the filter paper, then putting a container at 4 ℃ for cold storage, checking every 10 days, sowing the hybrid seeds to a special forest hole tray with the depth of 11cm after 75% of the hybrid seeds are exposed to the white, and performing insect killing and sterilization treatment on a substrate before sowing;
and 5: transplanting the hybrid seedling in the plug to a nursery garden when the hybrid seedling grows 4 true leaves and the root length is 10 cm; organic fertilizer is applied to nursery garden for 5.5m before transplanting3The method comprises the following steps of (1) deeply ploughing, leveling land, ridging and ridging, wherein the width of each furrow is 80cm, 4 rows of each furrow are planted, the width of each furrow is 20cm, the row spacing of each row is 20cm, the plant spacing is 17cm, the furrow spacing is 50cm, seedlings of the hybrid seedlings are thoroughly watered before being transplanted, a nursery garden is watered by sprinkling irrigation after being transplanted, 142 hybrid seedlings are survived after one month, topdressing is carried out from the middle ten days of 5 months to the middle ten days of 8 months in the same year, fertilizer is applied for 1 time every 10 days, 3 kg/mu of urea is applied every time, weeds are removed before topdressing, the height of the hybrid seedlings is 2.5m on average, and the land diameter is 1.2cm on average; the seedlings are easy to grow long branches and spines, are easy to scratch during operation, are not beneficial to inoculation operation in the future, branches and spines on the trunk are cut off in 8 months, and are cut again in the last ten days of 9 months;
step 6: inoculating rot germs in the last ten days of 8 months, selecting strains which are screened and have strong pathogenicity and wide distribution, preparing a PSA culture medium, sterilizing at the high temperature and the high pressure of 121 ℃ for 25min, subpackaging on an ultra-clean workbench by using an aseptic culture dish, inoculating the strains on the culture medium, culturing in an incubator at the temperature of 25 ℃, forming a fungus cake by using a puncher after 4d hyphae grow over the culture dish, cutting filter paper into squares of 2cm multiplied by 2cm, sterilizing at the high pressure for later use, preparing a preservative film, and cutting the preservative film into a width of 5 cm; inoculating hybrid seedlings with the ground diameter of more than 1cm, starting from the height of 80cm of the ground of the hybrid seedlings, inoculating a fungus cake every 15cm, namely, punching holes at the positions of 80cm, 95cm, 110cm, 125cm and 140cm of the main trunk of the hybrid seedlings by using a puncher respectively, wherein the hole depth touches a wood part, putting the back surfaces of the fungus cakes on three layers of filter paper which is completely soaked by sterile water, then completely corresponding one surfaces with hyphae to the holes on the main trunk of the hybrid seedlings, pressing the filter paper by hands to fix the filter paper, and then completely winding the filter paper by a preservative film from bottom to top to keep humidity;
and 7: investigation is carried out once after 3 weeks of inoculation, and the single plants which can see that the scab extends out of the preservative film are directly eliminated; second investigation is carried out after 4 weeks of inoculation, a preservative film is unfastened, filter paper is removed, whether each inoculation point is attacked and the length of disease spots is counted, and the morbidity of each single plant is calculated, wherein the morbidity is the incidence of the inoculation points/the total inoculation points multiplied by 100%; 2 plants with incidence rate of more than 70%, 43 plants with lesion length of more than 2cm and 43 plants with incidence rate of more than 70% and lesion length of more than 2cm in the hybrid combination; in the hybridization combination, 142 single plants are totally eliminated according to the standard of eliminating the single plants with the morbidity of more than 70% and the lesion length of more than 2cm, 43 single plants are totally eliminated, which accounts for 30% of the hybridization combination, and is equivalent to that 99 single plants enter a breeding garden to be screened in the next step, so that the cost is saved by at least 30%; the eliminated hybrid seedlings are cut off from the roots and then collected and burned in a centralized way;
and 8: and in spring of the next year, selecting the plumest bud within 10cm of the topmost end of the resistant single plant, and grafting the bud onto the pear rootstock with the planted plant-line spacing of 0.5m multiplied by 2 m.
TABLE 1 identification of rot resistance of New Gao X New Pear hybrid seedlings
| Incidence (%) | Plant number (strain) | Length of lesion (mm) | Plant number (strain) |
| The incidence rate is less than 10 | 30 | The length of the lesion is less than 10 | 25 |
| The disease rate is more than or equal to 10 and less than 20 | 73 | The length of the disease spot is more than or equal to 10 and less than 20 | 74 |
| The disease rate is more than or equal to 20 and less than 40 | 29 | 20 or more and the length of the lesion is less than 30 | 32 |
| The disease rate is more than or equal to 40 and less than 70 | 8 | The length of the disease spot is more than or equal to 30 and less than 40 | 8 |
| The incidence rate is more than or equal to 70 | 2 | The length of the lesion is more than or equal to 40 | 3 |
Example 2
A high-efficiency pear tree rot disease resistance breeding method comprises the following steps:
step 1: selecting a variety 'new high' with high rot disease as a female parent and a variety 'perfume' with high rot disease resistance as a male parent;
step 2: collecting flower buds in big balloon period, stripping off anthers, collecting, drying at 22 deg.C, loosening pollen, sealing at 4 deg.C for temporary storage, pollinating at big balloon period of female parent flower buds, bagging for isolation immediately after pollination for 10d, and removing isolation; bagging the fruits after 3 weeks of pollination, and spraying a solution of 800 times of thiophanate methyl and a solution of 4000 times of abamectin 5% once on the whole plant within 1 week before bagging; and soaking the bag opening with 500 times of 70% thiophanate methyl solution and 1000 times of chlorpyrifos the day before bagging; when the bag is sleeved, the bag opening is firstly propped up, the bag bottom is propped up, the bag body is expanded, and after the fruit is sleeved, the bag opening is sealed around the fruit handle; applying fruit strengthening fertilizer 5 months in the year, and applying 30kg of urea, 30kg of potassium sulfate and 4000kg of biogas slurry water per mu;
and step 3: harvesting the hybrid fruits which are physiologically mature, transporting the hybrid fruits back to the room after picking to allow the hybrid fruits to emit field heat, and then removing fruit bags; precooling for 24h at 20 ℃, and then storing in a refrigerator at 2 ℃; storing for 60 days, taking out the hybrid fruits from a refrigeration house, transversely cutting the hybrid fruits, taking out the hybrid seeds from ventricles by using tweezers, collecting the hybrid seeds by using seed bags, then scrubbing and washing the hybrid seeds at 25 ℃ in warm water, drying the seeds in the air after cleaning, and storing the seeds at room temperature;
and 4, step 4: the hybrid seeds are layered in the first 1 th of the year: firstly, putting hybrid seeds in warm water at 25 ℃ for fully absorbing water for 12 hours, additionally preparing a preservation box, putting filter paper with full water absorption at the bottom of the box, uniformly and non-overlapping the hybrid seeds on the filter paper, then putting a container at 0 ℃ for cold storage, checking every 10 days, sowing the hybrid seeds to a special forest hole tray with the depth of 11cm after 70% of the hybrid seeds are exposed to the white, and performing insect killing and sterilization treatment on a substrate before sowing;
and 5: transplanting the hybrid seedling in the plug to a nursery garden when the hybrid seedling grows 4 true leaves and the root length is 10 cm; organic fertilizer is applied for 4m in nursery before transplanting3Each mu of the plants is mixed with 30kg of urea, then the plants are deeply turned, the land is leveled, ridging and ridging are carried out, the width of each furrow is 80cm, 4 rows are planted in one furrow, and the plants are planted in wide and narrow rowsPlanting, wherein two narrow rows and one wide row are arranged in each furrow to facilitate operations such as late inoculation and sprout removal, the row spacing of the wide rows is 40cm, the row spacing of the narrow rows is 20cm, the plant spacing is 15cm, the furrow spacing is 50cm, the seedlings of the hybrid seedlings before transplanting are fully watered thoroughly, the nursery garden after transplanting is watered by sprinkling irrigation, 896 hybrid seedlings survive after one month are obtained, topdressing is carried out from 5 middle ten days to 8 middle ten days of the year, fertilizer is applied for 1 time every 10 days, 2 kg/mu of urea is applied every time, weeds are removed before topdressing, the height of the hybrid seedlings is 3m on average, and the ground diameter is 1.1cm on average; the seedlings are easy to grow long branches and spines, are easy to scratch during operation, are not beneficial to inoculation operation in the future, branches and spines on the trunk are cut off in 8 months, and are cut again in the last ten days of 9 months;
step 6: inoculating rot germs in the last ten days of 8 months, selecting strains which are screened and have strong pathogenicity and wide distribution, preparing a PSA culture medium, sterilizing at the high temperature and the high pressure of 121 ℃ for 25min, subpackaging on an ultra-clean workbench by using an aseptic culture dish, inoculating the strains on the culture medium, culturing in an incubator at the temperature of 25 ℃, forming a fungus cake by using a puncher after 4d hyphae grow over the culture dish, cutting filter paper into squares of 2cm multiplied by 2cm, sterilizing at the high pressure for later use, preparing a preservative film, and cutting the preservative film into a width of 5 cm; inoculating hybrid seedlings with the ground diameter of more than 1cm, starting from the height of 80cm of the ground of the hybrid seedlings, inoculating a fungus cake every 15cm, namely, punching holes at the positions of 80cm, 95cm, 110cm, 125cm and 140cm of the main trunk of the hybrid seedlings by using a puncher respectively, wherein the hole depth touches a wood part, putting the back surfaces of the fungus cakes on three layers of filter paper which is completely soaked by sterile water, then completely corresponding one surfaces with hyphae to the holes on the main trunk of the hybrid seedlings, pressing the filter paper by hands to fix the filter paper, and then completely winding the filter paper by a preservative film from bottom to top to keep humidity;
and 7: investigation is carried out once after 3 weeks of inoculation, and the single plants which can see that the scab extends out of the preservative film are directly eliminated; second investigation is carried out after 4 weeks of inoculation, a preservative film is unfastened, filter paper is removed, whether each inoculation point is attacked and the length of disease spots is counted, and the morbidity of each single plant is calculated, wherein the morbidity is the incidence of the inoculation points/the total inoculation points multiplied by 100%; 400 plants with incidence rate of more than 40 percent, 297 plants with lesion length of more than 2cm and 453 plants with incidence rate of more than 40 percent and lesion length of more than 2cm in total in the hybrid combination; in the hybridization combination, 896 single plants are totally eliminated, 453 single plants account for 51 percent of the hybridization combination according to the standard of eliminating single plants with the incidence rate of more than 40 percent and the lesion length of more than 2cm, 443 single plants are subjected to next screening, and 443 single plants can meet the requirements of subsequent breeding for the breeding of pear trees; the eliminated hybrid seedlings are cut off from the roots and then collected and burned in a centralized way;
89 strains with the incidence rate of more than 70 percent, 297 strains with the lesion length of more than 2cm and 297 strains with the incidence rate of more than 70 percent and the lesion length of more than 2cm in the hybridization combination are counted, if the exclusion 297 strains account for 33 percent of the hybridization combination according to the standard of eliminating the single strains with the incidence rate of more than 70 percent and the lesion length of more than 2cm, 599 strains are subjected to the next screening. Under two elimination conditions, the method for eliminating the single plant with the morbidity of more than 40% and the length of the disease spot of more than 2cm is more than 156 plants compared with the method for eliminating the single plant with the morbidity of more than 70% and the length of the disease spot of more than 2cm, and the land can be saved by 1 mu by only one combination;
and 8: and in spring of the next year, selecting the plumest bud within 10cm of the topmost end of the resistant single plant, and grafting the bud onto the pear rootstock with the planted plant-line spacing of 0.5m multiplied by 2 m.
TABLE 2 identification of rot disease resistance of new Gao X perfume hybrid seedlings
| Incidence (%) | Plant number (strain) | Length of lesion (mm) | Plant number (strain) |
| The incidence rate is less than 10 | 99 | The length of the lesion is less than 10 | 241 |
| The disease rate is more than or equal to 10 and less than 20 | 163 | The length of the disease spot is more than or equal to 10 and less than 20 | 358 |
| The disease rate is more than or equal to 20 and less than 40 | 234 | 20 or more and the length of the lesion is less than 30 | 145 |
| The disease rate is more than or equal to 40 and less than 70 | 311 | The length of the disease spot is more than or equal to 30 and less than 40 | 100 |
| The incidence rate is more than or equal to 70 | 89 | The length of the lesion is more than or equal to 40 | 52 |
Example 3
A high-efficiency pear tree rot disease resistance breeding method comprises the following steps:
step 1: selecting a variety 'early red koumiss' with rot disease as a female parent and a variety 'late beautiful pear' with high rot disease as a male parent;
step 2: collecting flower buds in big balloon period, stripping off anthers, collecting, drying at 24 deg.C, loosening pollen, sealing at 4 deg.C for temporary storage, pollinating at big balloon period of female parent flower buds, bagging for isolation immediately after pollination, and removing isolation after pollination for 12 d; bagging the fruits after 3 weeks of pollination, and spraying a solution of 800 times of thiophanate methyl and a solution of 4000 times of abamectin 5% once on the whole plant within 1 week before bagging; and soaking the bag opening with 500 times of 70% thiophanate methyl solution and 1000 times of chlorpyrifos the day before bagging; when the bag is sleeved, the bag opening is firstly propped up, the bag bottom is propped up, the bag body is expanded, and after the fruit is sleeved, the bag opening is sealed around the fruit handle; applying fruit strengthening fertilizer 5 months in the year, and applying 30kg of urea, 30kg of potassium sulfate and 4000kg of biogas slurry water per mu;
and step 3: harvesting the hybrid fruits which are physiologically mature, transporting the hybrid fruits back to the room after picking to allow the hybrid fruits to emit field heat, and then removing fruit bags; precooling for 24h at 17 ℃, and then storing in a refrigerator at 1 ℃; after 80 days of storage, taking the hybrid fruits out of a refrigeration house, transversely cutting the hybrid fruits, taking the hybrid seeds out of ventricles by using tweezers, collecting the hybrid seeds by using seed bags, then scrubbing the hybrid seeds at 25 ℃ in warm water, drying the seeds in the air after cleaning the seeds, and storing the seeds at room temperature;
and 4, step 4: the hybrid seeds are layered in 12 last ten days of the year: firstly, putting hybrid seeds in warm water at 25 ℃ for fully absorbing water for 12 hours, additionally preparing a preservation box, putting filter paper with full water absorption at the bottom of the box, uniformly and non-overlapping the hybrid seeds on the filter paper, then putting the container at 2 ℃ for cold storage, checking every 10 days, sowing the hybrid seeds to a special forest hole tray with the depth of 11cm after 80% of the hybrid seeds are exposed to the white, and performing insect killing and sterilization treatment on a substrate before sowing;
and 5: transplanting the hybrid seedling in the plug to a nursery garden when the hybrid seedling grows 4 true leaves and the root length is 10 cm; organic fertilizer is applied to 7m in nursery before transplanting3The method comprises the following steps of (1) deeply ploughing, leveling land, ridging and ridging, wherein the width of each furrow is 80cm, 4 rows of each furrow are planted, the width of each furrow is 20cm, the row spacing of each furrow is 40cm, the row spacing of each narrow row is 20cm, the plant spacing is 20cm, the furrow spacing is 50cm, seedlings of the hybrid seedlings before transplanting are fully watered thoroughly, a nursery garden after transplanting is watered by sprinkling irrigation, 137 hybrid seedlings survive after one month, topdressing is carried out from the middle ten days of the 5 th month to the middle ten days of the 8 th month in the same year, fertilizer application is carried out for 1 time every 10d, 4 kg/mu of urea is applied each time, weeds are removed before topdressing, the height of the hybrid seedlings is average 2.8m, and the land diameter is average 1.15 cm; the seedlings are easy to have long branches and thorns, are easy to scratch during the operation, are not beneficial to the future inoculation operation, and will grow in 8 monthsCutting off branches and thorns on the trunk, and cutting again in the last ten days of 9 months;
step 6: inoculating rot germs in the last ten days of 8 months, selecting strains which are screened and have strong pathogenicity and wide distribution, preparing a PSA culture medium, sterilizing at the high temperature and the high pressure of 121 ℃ for 25min, subpackaging on an ultra-clean workbench by using an aseptic culture dish, inoculating the strains on the culture medium, culturing in an incubator at the temperature of 25 ℃, forming a fungus cake by using a puncher after 4d hyphae grow over the culture dish, cutting filter paper into squares of 2cm multiplied by 2cm, sterilizing at the high pressure for later use, preparing a preservative film, and cutting the preservative film into a width of 5 cm; inoculating hybrid seedlings with the ground diameter of more than 1cm, starting from the height of 80cm of the ground of the hybrid seedlings, inoculating a fungus cake every 15cm, namely, punching holes at the positions of 80cm, 95cm, 110cm, 125cm and 140cm of the main trunk of the hybrid seedlings by using a puncher respectively, wherein the hole depth touches a wood part, putting the back surfaces of the fungus cakes on three layers of filter paper which is completely soaked by sterile water, then completely corresponding one surfaces with hyphae to the holes on the main trunk of the hybrid seedlings, pressing the filter paper by hands to fix the filter paper, and then completely winding the filter paper by a preservative film from bottom to top to keep humidity;
and 7: investigation is carried out once after 3 weeks of inoculation, and the single plants which can see that the scab extends out of the preservative film are directly eliminated; second investigation is carried out after 4 weeks of inoculation, a preservative film is unfastened, filter paper is removed, whether each inoculation point is attacked and the length of disease spots is counted, and the morbidity of each single plant is calculated, wherein the morbidity is the incidence of the inoculation points/the total inoculation points multiplied by 100%; 59 plants with the incidence rate of more than 70 percent, 77 plants with the lesion length of more than 2cm and a total of 82 plants with the incidence rate of more than 70 percent and the lesion length of more than 2cm in the hybrid combination; the hybridization combination totals 137 strains, and 82 strains are eliminated altogether according to the standard of eliminating the single strains with the morbidity of more than 70 percent and the lesion length of more than 2cm, which accounts for 60 percent of the hybridization combination. The eliminated hybrid seedlings are cut off from the roots and then collected and burned in a centralized way; because the combined parents are all susceptible varieties, the disease resistance of single plants in the offspring is poor, 60 percent of the plants are directly eliminated in the seedling stage, which is equivalent to that only 55 single plants enter a breeding garden to be screened in the next step. The resistance of the parent variety has great influence on the number of resistant plants of the offspring, both parents are not resistant varieties in the embodiment, the proportion of disease-resistant plants in filial generations is small, and the breeding work is not facilitated for further breeding the variety which is disease-resistant and has high fruit quality;
and 8: and in spring of the next year, selecting the plumest bud within 10cm of the topmost end of the resistant single plant, and grafting the bud onto the pear rootstock with the planted plant-line spacing of 0.5m multiplied by 2 m.
TABLE 3 early Red Coomassie X late beautiful pear hybrid rot disease resistance identification
| Incidence (%) | Plant number (strain) | Length of lesion (mm) | Plant number (strain) |
| The incidence rate is less than 10 | 24 | The length of the lesion is less than 10 | 24 |
| The disease rate is more than or equal to 10 and less than 20 | 0 | The length of the disease spot is more than or equal to 10 and less than 20 | 36 |
| The disease rate is more than or equal to 20 and less than 40 | 25 | 20 or more and the length of the lesion is less than 30 | 65 |
| The disease rate is more than or equal to 40 and less than 70 | 29 | The length of the disease spot is more than or equal to 30 and less than 40 | 6 |
| The incidence rate is more than or equal to 70 | 59 | The length of the lesion is more than or equal to 40 | 6 |
The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily implemented by those skilled in the art by means of replacement or modification according to the technical contents disclosed in the specification, and therefore, all changes and modifications that come within the spirit and technical conditions of the present invention should be included in the claims of the present invention.
Claims (7)
1. A high-efficiency pear tree rot disease resistance breeding method is characterized by comprising the following steps:
step 1: selecting a hybrid combination in which at least one parent has rot disease resistance to be more than resistant;
step 2: collecting buds of the male parent in a big balloon period, peeling off anthers, drying, keeping at the drying temperature of 22-25 ℃, sealing and storing at 4 ℃ after pollen scattering, pollinating the buds of the female parent in the big balloon period, immediately bagging and isolating, and removing the isolation after pollination for 10-14 days; bagging the fruits after 3 weeks of pollination, spraying the pesticide and the bactericide on the whole plant once in 1 week before bagging, and soaking the bag opening with 500 times of 70% thiophanate methyl solution and 1000 times of chlorpyrifos solution before bagging;
and step 3: harvesting the hybrid fruits which are physiologically mature, transporting the hybrid fruits back to the room to emit field heat, and then removing fruit bags; pre-cooling for 24 hours at the temperature of 15-20 ℃, then storing in a cold storage at the temperature of 0-2 ℃ for 60-80 days, taking out the hybrid seeds, scrubbing with warm water at the temperature of 25 ℃, then drying in the air, and storing at room temperature;
and 4, step 4: and (3) stacking hybrid seeds in the last 12 th or the last 1 st of the year: firstly, soaking hybrid seeds in warm water at 25 ℃ for 12 hours, then uniformly placing the hybrid seeds in a container with water-absorbing filter paper at the bottom, refrigerating the container at 0-4 ℃, keeping the filter paper moist, and sowing the hybrid seeds in a special hole tray for forest trees with the depth of 11cm after 70-80% of the hybrid seeds are exposed to the white;
and 5: when 4 true leaves and root length of the hybrid seedling are 10cm, transplanting the hybrid seedling to a nursery land with the furrow width of 80cm, planting 4 rows in one furrow and planting in wide and narrow rows; cutting off branches and thorns on the trunk in 8 months of the transplanting current year, and re-cutting once in late 9 months;
step 6: and (3) inoculating pear tree canker in 8 last ten days of the year: firstly, culturing pear rot germs in an incubator at 25 ℃ for 4 days, then beating hyphae into fungus cakes by using a puncher, inoculating hybrid seedlings with the ground diameter of more than 1cm, and inoculating five fungus cakes every 15cm upwards from the ground height of 80cm of the hybrid seedlings during inoculation; the inoculation method comprises the steps of punching holes with a puncher at positions 80cm, 95cm, 110cm, 125cm and 140cm high of the main stem of the hybrid seedling, enabling the depth of the holes to touch the xylem, placing the back surface of a fungus cake on three layers of filter paper or absorbent cotton which is completely soaked by sterile water, enabling the surface with hyphae to completely correspond to the holes in the main stem of the hybrid seedling, and then completely winding the main stem of the hybrid seedling from bottom to top by using a preservative film; the pear tree rot disease bacteria culture medium is a PSA culture medium;
and 7: after 3 weeks of inoculation, the single plants with the scabs extending out of the preservative film are eliminated; after 4 weeks of inoculation, the number of single plants in the hybrid combination is more than 500, and single plants with the morbidity of more than 40 percent and the lesion length of more than 2cm are eliminated; the number of single plants in the hybrid combination is less than 500, and the single plants with the morbidity of more than 70 percent and the lesion length of more than 2cm are eliminated;
and 8: and in spring of the next year, selecting the plumest bud within 10cm of the topmost end of the resistant single plant, and grafting the bud onto the pear rootstock with the planted plant-line spacing of 0.5m multiplied by 2 m.
2. The method of claim 1, further comprising applying strong fruit fertilizer in 5 months of the year of hybridization with 30kg of urea, 30kg of potassium sulfate and 4000kg of biogas slurry water per acre.
3. The method of claim 1, wherein the drying temperature in step 2 is 25 ℃; the segregation was removed after pollination for 14 d.
4. The method according to claim 1, wherein the pesticide in step 2 is 5% 4000 times liquid abamectin, and the bactericide is 70% 800 times liquid thiophanate methyl.
5. The method of claim 1, wherein the pre-cooling temperature in step 3 is 15 ℃, the storage temperature is 0 ℃, and the storage time is 70 d.
6. The method of claim 1, further comprising performing high-pressure organic fertilizer topdressing at 4-7 m/mu and urea 30 kg/mu in the nursery before transplanting the hybrid seedlings in step 5; and (3) thoroughly watering the hybrid seedlings before transplanting, carrying out spray irrigation and watering on the transplanted seedlings in a nursery garden, topdressing from 5 middle ten days to 8 middle ten days of the year, fertilizing 1 time every 10 days, and applying 2-4 kg/mu of urea every time.
7. The method according to claim 1, further comprising the step 5 of arranging 2 narrow rows and 1 wide row per furrow, wherein the row spacing of the wide rows is 40cm, the row spacing of the narrow rows is 20cm, the plant spacing is 15-20 cm, and the furrow spacing is 50 cm.
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