CN108277165B - Wheat scab grain strain production and use method - Google Patents

Abstract

The invention relates to the cultivation of plant fungus disease pathogenic bacteria, in particular to a method for manufacturing and using wheat scab grain strains, solving the problems that the prior inoculation identification method needs to culture a bacterial solution, the method is complex, the bacterial solution can not be stored for a long time, and the inoculation success rate is low, and preparing the grain strains: soaking 5% mung bean water in the shelled grains, keeping water retention rate at 60%, packaging into bags, sealing, sterilizing at high temperature and high pressure, air drying to room temperature, culturing with dark bacteria at both ends under constant temperature and high humidity, transferring to air-permeable paper bags, and keeping the sealed bags at low temperature and in dark place. The using method comprises the following steps: the activity is detected firstly, the young palea is inoculated on the young side of the base part before blooming after ear emergence, the palea is wrapped and moisturized until the palea turns brown, the wrapping layer is taken off, and the palea is moisturized for 28 days in high humidity mist. The advantages are that: 1. the strain can be preserved for a long time; 2. the product is manufactured once, is used for many times, has small dosage and is convenient to transport; 3. the inoculation method is simple and efficient, the morbidity is high, and the disease resistance identification result is accurate.

Description

Wheat scab grain strain production and use method

Technical Field

The invention relates to the cultivation of plant fungal disease pathogenic bacteria, in particular to a method for preparing and using wheat scab grain strains.

Background

Wheat scab (FHB) is a devastating disease caused by Fusarium graminearum that seriously damages wheat ears and grains, and mainly occurs in humid and semi-humid areas such as the middle and lower reaches of the Yangtze river, winter wheat areas in the river basin of Huai river, and spring wheat areas in the northeast of China. The disease can cause 70 to 100 percent of yield loss of wheat in epidemic years, and the wheat grains infected by gibberella contains various toxins, particularly DON toxin, has strong toxicity to people and animals, and can cause vomiting, diarrhea, skin irritation, food refusal, nervous disorder, abortion, stillbirth and the like of the people and the animals. When the wheat disease rate reaches more than 4 percent, the wheat can not be eaten, and the commodity value is lost.

In recent years, along with the global warming aggravation and the change of cultivation systems, early nino and lanina events alternately occur, extreme weather frequently occurs, wheat scab gradually rises from accidental diseases to frequent diseases, the occurrence area is continuously enlarged, the scab is expanded from frequent middle and lower wheat areas of Yangtze river to Huang-Huai-Mai areas and Huabei-Mai areas, and the wheat areas of Huai-river basin are one of the areas with the most serious scab damage. However, no effective method for preventing and controlling the gibberellic disease exists in the world, and no chemical agent capable of thoroughly preventing and controlling the gibberellic disease of wheat is developed, so that the breeding and popularization of the scab-resistant variety becomes the most economic and environment-friendly way for controlling and preventing the gibberellic disease of wheat at present.

Inoculation identification is an important means for breeding wheat scab-resistant varieties, common methods include a single flower drip method, a bacterial liquid spraying method, an earth surface inoculation method and the like, and the methods have advantages and disadvantages respectively. The inoculation is accurate by the single flower drip method, the inoculation success rate can be ensured, and qualitative and quantitative identification can be carried out; however, before inoculation, bacterial liquid culture is required, the required equipment is complex, the requirements on environment and culture conditions are strict, the number of viable bacteria needs to be detected under a microscope before each inoculation, and the cultured bacterial liquid cannot be stored for a long time and is easy to lose activity, so that the identification of the gibberellic disease is very inconvenient. The bacterium liquid spraying method is convenient for bacterium inoculation, and is beneficial to large-area and large-scale bacterium inoculation identification; however, the bacterial liquid needs to be cultured before each inoculation, and the number of viable bacteria is detected under a microscope, so that the bacterial liquid is inconvenient to store, the storage time is short, and the inoculation success rate is low. The soil surface inoculation method adopts cereal grains to culture strains, the strains are sprayed on the soil surface and then naturally attack by spore ejection, the requirement on the environment is high, the inoculation success rate is low, and qualitative and quantitative identification on wheat varieties is difficult.

Therefore, it is necessary to research wheat scab grains which are simple in preparation and use methods, high in inoculation success rate and capable of being stored for a long time and use methods.

Disclosure of Invention

The invention solves the problems that the existing bacterium inoculation identification method needs to culture a bacterium solution, the preparation method is complex, the cultured bacterium solution cannot be stored for a long time, and the success rate of bacterium inoculation is low, and provides a method for preparing and using a wheat scab grain strain, which has the advantages of simple preparation method, no need of bacterium solution culture, long-term preservation of prepared grains without losing activity, and high success rate of bacterium inoculation.

The invention is realized by the following operation steps: the method for preparing the wheat scab grain strain comprises the following operation steps:

a. preparing fresh and full mung beans which are not damaged by worms and are moldy, adding 1200ml of purified water, boiling on fire, boiling the water for 15-20min, turning off the fire when the mung beans just bloom, filtering mung bean residues with fine gauze, only keeping the mung bean water, supplementing the purified water to 1000ml, and preparing the mung bean water with the concentration of 5% for later use;

b. preparing 1kg of fresh shelled grains which are dry, have no mildew, and have no clean floating skin and broken slag, putting the grains into a clean container, adding warm 5% mung bean water, fully stirring to completely wet the grains, covering the container and keeping the moisture for 4 hours to ensure that the grains fully absorb water;

c. filtering to remove excessive water, spreading the soaked grains in a cool and ventilated place, air drying for 30min to make the water holding rate of the grains reach about 60%, and kneading with hand to obtain dough;

d. subpackaging the treated grains into high-temperature and high-pressure resistant polypropylene sterilization bags, wherein each bag is subpackaged with 300-;

e. sealing two ends of the high-temperature and high-pressure resistant polypropylene sterilization bag by using a breathable strain bag sponge sealing lantern ring to manufacture a strain cylinder, so as to prevent mixed bacteria pollution;

f. placing the prepared fungus cylinders in an autoclave, sterilizing at 125 ℃ for 2 hours under high temperature and high pressure, and airing to room temperature after air release;

g. opening a sponge cover of a sponge sealing lantern ring at one end of the bacteria cylinder on a super-clean workbench, clamping a small piece of large soybean trichoderma strain by using sterilized tweezers, putting the small piece of large soybean strain on grains at one end of the bacteria cylinder, and covering a breathable sponge cover; processing the other end by the method to make the two ends of the fungus cylinder connected with the strains;

h. placing the fungus cylinders inoculated with the fungus in a completely dark incubator or culture room for constant temperature culture at 25 ℃, keeping ventilation, keeping the relative humidity of the incubator or culture room about 60%, turning over the fungus cylinders once every 1-2 days during the culture period of the fungus to ensure the growth of the hyphae to be consistent, about 7-10 days, and finishing the culture of the fungus cylinders after the hyphae grow over the fungus cylinders;

i. opening one end of the cultured fungus cylinder on a superclean bench, transferring the strain into a sterile breathable paper bag, pressing grains with the fungus to disperse, and hanging and airing the grains in a cool and ventilated place;

j. the completely dried strain can be put into a sterile self-sealing plastic bag, a strain label is attached, the name and the date of the strain are noted, and the strain is stored in a dark place at the temperature of 4 ℃; or vacuum packaging, vacuumizing, and storing at-20 deg.C for a long time.

The application method of the wheat scab grain strain comprises the following operation steps:

firstly, detecting activity: laying a piece of filter paper in a clean culture dish, adding a small amount of sterilized distilled water to completely wet the filter paper, putting 50 grains of strains in each culture dish, completely dispersing the strains, covering the strains, sticking strain labels, placing the strains in a completely dark culture box for constant temperature culture at 25 ℃, observing the growth condition of the strains after 36 hours, indicating that the strains have activity if white strains grow all around the grains of the strains, indicating that the strains lose activity if the grains of the strains do not grow the strains, arranging for 2-3 times of repetition, recording and counting the proportion of the strains with the activity, and if the strains with the activity account for more than 90 percent, using the strains for field inoculation;

inoculating by adopting a single-flower inoculation method, wherein the inoculation period is from after ear emergence of the wheat to before flowering, the inoculation part is the right-side floret of the 6 th floret with the lowest side of the wheat base part and the upward side of the floret, selecting ears with 2-3 days of ear emergence, clamping a grain strain by using a forceps, placing the grain strain into the inner shell of the right-side floret of the target floret, spraying 2-3 times of purified water by using a handheld spraying kettle aiming at the ear of the bacteria, keeping the ears moist, wrapping the ears with a layer of transparent preservative film, or covering the ears with a PE plastic bag for moisturizing, hanging a label, and recording the inoculation time, wherein at least 10 ears are inoculated on each row of materials;

observing the inoculated part after inoculation, keeping the moisture for about 3-5 days, and taking down a preservative film or a plastic bag after the palea of the inoculated spikelet turns brown;

fourthly, a mist moisturizing system is additionally arranged, water is sprayed for 5-10 times a day, so that the growth environment keeps the humidity more than 50%, the higher the humidity is, the more the gibberellic fungus is expanded, and if the climate is dry, the water spraying times are properly increased;

and fifthly, investigating the scab incidence condition 21 days and 28 days after inoculation, wherein the investigation method adopts a conventional method for investigation.

Compared with the prior art, the wheat scab grain strain has the following advantages: 1. compared with the single flower drip method: the single flower drop method needs to use a constant-temperature shaking table to culture the bacterial liquid, the number of spores needs to be detected under a microscope before each inoculation, the cultured bacterial liquid cannot be stored for a long time (the cultured bacterial liquid cannot be stored for more than 1 month under the condition of 4 ℃), the spores are easy to lose activity, and the identification and carrying at different places are inconvenient; after the grain strain prepared by the invention uses 5 percent of mung bean water to soak the grain, the nutrient content is rich, which is not only beneficial to the growth of the gibberella hyphae trophosome, but also beneficial to the formation of gibberella spores, and does not need a constant temperature shaking table, only needs to be cultured for 7-10 days under the constant temperature condition of about 25 ℃ (the culture time is longer because of the large amount of the breeding bacteria), the culture condition is easier to realize compared with the green bean aqueous liquid culture, the dried grain with bacteria and the gibberella spores are in the dormant state, which is beneficial to the long-term storage of the strains and is convenient to carry to different places for inoculation and identification, and the spore is quickly germinated and the vegetative growth of the grain strain is recovered under the condition of proper temperature and humidity, and experiments show that the strain preserved at the temperature of 4 ℃ and the strain preserved at the temperature of-20 ℃ have the activity of over 95 percent after being preserved for 6 months. 2. Compared with a bacterial liquid spraying method: the bacteria liquid spraying method is suitable for large-area and large-scale inoculation identification, but is greatly influenced by the environment, the success rate of inoculation is low, the identification target cannot be accurately, qualitatively and quantitatively analyzed, and the method is only suitable for disease-resistant identification of bred varieties and is not suitable for identification of genetic group single plants (or single ears); the grain strain produced by the invention can be used for single plant inoculation, can ensure the inoculation effect and the disease success rate, and is suitable for single plant (or single spike) identification of genetic groups. 3. Compared with the soil surface inoculation method: the soil surface inoculation method usually adopts grain seeds (such as wheat, corn, rice grains and the like) to culture strains, the strains are sprayed on the soil surface and then naturally attack by spore ejection, the requirement on the environment is high, the inoculation success rate is low, and the qualitative and quantitative identification of wheat varieties is difficult; the grain strain prepared by the invention has small single grain size, the grain is shelled, the strain is not easy to adhere in the strain preparation process, the adhesion generated by hypha growth is easy to disperse into single grain, the grain can be scattered on the soil surface for soil surface inoculation after being dried, the single grain can be clamped by tweezers and placed on the palea of the wheat ear floret, the same inoculation effect as the single flower drip method is achieved, and the identification of the genetic group single plant (or single ear) can be carried out, therefore, the application range is wider than that of the strain prepared by wheat, corn, rice grains and the like. 4. In addition, the grain strain prepared by the invention is prepared once and used for many times, the grain is small, the weight of each thousand grains is only about 2.2-4.0g, 1kg of strain is prepared once, 22-40 ten thousand grains can be obtained, namely 22-40 ten thousand wheat ears can be inoculated, the use amount is small, the transportation is convenient, and the strain prepared once can be used for many times; 5. the inoculation method is simple and efficient, has high morbidity and accurate disease resistance identification result, and can completely replace the traditional single flower drip method for identifying the gibberellic disease resistance.

Detailed Description

The method for preparing the wheat scab grain strain comprises the following operation steps:

a. preparing fresh and full mung beans which are not damaged by worms and are moldy, adding 1200ml of purified water, boiling on fire, boiling the water for 15-20min, turning off the fire when the mung beans just bloom, filtering mung bean residues with fine gauze, only keeping the mung bean water, supplementing the purified water to 1000ml, and preparing the mung bean water with the concentration of 5% for later use;

b. preparing 1kg of fresh shelled grains which are dry, have no mildew, and have no clean floating skin and broken slag, putting the grains into a clean container, adding warm 5% mung bean water, fully stirring to completely wet the grains, covering the container and keeping the moisture for 4 hours to ensure that the grains fully absorb water;

c. filtering to remove excessive water, spreading the soaked grains in a cool and ventilated place, air drying for 30min to make the water holding rate of the grains reach about 60%, and kneading with hand to obtain dough;

d. subpackaging the treated grains into high-temperature and high-pressure resistant polypropylene sterilization bags, wherein each bag is subpackaged with 300-;

e. sealing two ends of the high-temperature and high-pressure resistant polypropylene sterilization bag by using a breathable strain bag sponge sealing lantern ring to manufacture a strain cylinder, so as to prevent mixed bacteria pollution;

f. placing the prepared fungus cylinders in an autoclave, sterilizing at 125 ℃ for 2 hours under high temperature and high pressure, and airing to room temperature after air release;

g. opening a sponge cover of a sponge sealing lantern ring at one end of the bacteria cylinder on a super-clean workbench, clamping a small piece of large soybean trichoderma strain by using sterilized tweezers, putting the small piece of large soybean strain on grains at one end of the bacteria cylinder, and covering a breathable sponge cover; processing the other end by the method to make the two ends of the fungus cylinder connected with the strains;

h. placing the fungus cylinders inoculated with the fungus in a completely dark incubator or culture room for constant temperature culture at 25 ℃, keeping ventilation, keeping the relative humidity of the incubator or culture room about 60%, turning over the fungus cylinders once every 1-2 days during the culture period of the fungus to ensure the growth of the hyphae to be consistent, about 7-10 days, and finishing the culture of the fungus cylinders after the hyphae grow over the fungus cylinders;

i. opening one end of the cultured fungus cylinder on a superclean bench, transferring the strain into a sterile breathable paper bag, pressing grains with the fungus to disperse, and hanging and airing the grains in a cool and ventilated place;

j. the completely dried strain can be put into a sterile self-sealing plastic bag, a strain label is attached, the name and the date of the strain are noted, and the strain is stored in a dark place at the temperature of 4 ℃; or vacuum packaging, vacuumizing, and storing at-20 deg.C for a long time.

The application method of the wheat scab grain strain comprises the following operation steps:

firstly, detecting activity: laying a piece of filter paper in a clean culture dish, adding a small amount of sterilized distilled water to completely wet the filter paper, putting 50 grains of strains in each culture dish, completely dispersing the strains, covering the strains, sticking strain labels, placing the strains in a completely dark culture box for constant temperature culture at 25 ℃, observing the growth condition of the strains after 36 hours, indicating that the strains have activity if white strains grow all around the grains of the strains, indicating that the strains lose activity if the grains of the strains do not grow the strains, arranging for 2-3 times of repetition, recording and counting the proportion of the strains with the activity, and if the strains with the activity account for more than 90 percent, using the strains for field inoculation;

inoculating by adopting a single-flower inoculation method, wherein the inoculation period is from after ear emergence of the wheat to before flowering, the inoculation part is the right-side floret of the 6 th floret with the lowest side of the wheat base part and the upward side of the floret, selecting ears with 2-3 days of ear emergence, clamping a grain strain by using a forceps, placing the grain strain into the inner shell of the right-side floret of the target floret, spraying 2-3 times of purified water by using a handheld spraying kettle aiming at the ear of the bacteria, keeping the ears moist, wrapping the ears with a layer of transparent preservative film, or covering the ears with a PE plastic bag for moisturizing, hanging a label, and recording the inoculation time, wherein at least 10 ears are inoculated on each row of materials;

observing the inoculated part after inoculation, keeping the moisture for about 3-5 days, and taking down a preservative film or a plastic bag after the palea of the inoculated spikelet turns brown;

fourthly, a mist moisturizing system is additionally arranged, water is sprayed for 5-10 times a day, so that the growth environment keeps the humidity more than 50%, the higher the humidity is, the more the gibberellic fungus is expanded, and if the climate is dry, the water spraying times are properly increased;

and fifthly, investigating the scab incidence condition 21 days and 28 days after inoculation, wherein the investigation method adopts a conventional method for investigation.

The first embodiment is as follows: in a wheat test field in a sunlight greenhouse of a scientific and technological innovation base of agricultural academy of sciences in certain province and city in the north in 2017, 695 ear inoculations of 139 rows of wheat are identified by using the wheat gibberella grain strains prepared by the method. Wherein 12 spikes are broken due to human reasons, and the identification is invalid. In the rest 683 spikes, 671 effective pathogenic spikes are found, and the incidence rate of inoculation is 98.24%. In total, 35 new wheat lines with high resistance to immune head blight are identified, wherein the ear disease rate of 6 lines is 5.48%, and the wheat line with the highest resistance is 139 lines. The disease spikelet rate of the susceptible control variety Mianyang 8545 is 55.56%, and the disease spikelet rate of the disease-resistant control Sumai No. 3 is 10.01%.

Example two: in the same year of 4 months, 1200 ear inoculations of 120 rows of wheat are identified by using the wheat gibberella grain strains prepared by the method in a wheat test field in a modern agriculture science and technology innovation demonstration garden of agricultural academy of sciences of certain province and city in the south. 39 spikes among them drop off the moisture retention film due to wind blowing, rain and other reasons, and the identification is invalid. The effective outbreak ears of the rest 1161 ears are 1093, and the inoculation morbidity is 94.14%. 27 new wheat lines with high resistance to immune head blight are identified in total, wherein the ear disease rate of 8 lines is less than 6%, and the resistance of all inoculated ears in the whole row is consistent. The disease spikelet rate of the susceptible control variety Mianyang 8545 is 48.22%, the disease spikelet rate of the susceptible control Alondra is 57.64%, the disease spikelet rate of the disease-resistant control Sumai No. 3 is 9.26%, and the disease spikelet rate of the disease-resistant control expecting water white is 11.38%.

Claims (2)

1. A method for preparing wheat scab grain strains comprises the following operation steps:

a. preparing fresh and full mung beans which are not damaged by worms and are moldy, adding 1200ml of purified water, boiling on fire, boiling the water for 15-20min, turning off the fire when the mung beans just bloom, filtering mung bean residues with fine gauze, only keeping the mung bean water, supplementing the purified water to 1000ml, and preparing the mung bean water with the concentration of 5% for later use;

b. preparing 1kg of fresh shelled grains which are dry, have no mildew, and have no clean floating skin and broken slag, putting the grains into a clean container, adding warm 5% mung bean water, fully stirring to completely wet the grains, covering the container and keeping the moisture for 4 hours to ensure that the grains fully absorb water;

c. filtering to remove excessive water, spreading the soaked grains in a cool and ventilated place, air drying for 30min to make the water holding rate of the grains reach 60%, and kneading with hand to obtain the final product;

d. subpackaging the treated grains into polypropylene sterilization bags, wherein each bag is subpackaged with 300-;

e. sealing two ends of the polypropylene sterilization bag by using a breathable strain bag sponge sealing lantern ring to manufacture a strain cylinder, so as to prevent mixed bacteria pollution;

f. placing the prepared fungus cylinders in an autoclave, sterilizing for 2 hours at 125 ℃, deflating and airing to room temperature;

g. opening a sponge cover of a sponge sealing lantern ring at one end of the bacteria cylinder on a super-clean workbench, clamping a small piece of large soybean trichoderma strain by using sterilized tweezers, putting the small piece of large soybean strain on grains at one end of the bacteria cylinder, and covering a breathable sponge cover; processing the other end by the method to make the two ends of the fungus cylinder connected with the strains;

h. placing the fungus cylinders inoculated with the fungus in a completely dark incubator or culture room for constant temperature culture at 25 ℃, keeping ventilation, keeping the relative humidity of the incubator or culture room at 60%, turning over the fungus cylinders once every 1-2 days during the culture period of the fungus to ensure the growth of the hyphae to be consistent, and finishing the culture of the fungus cylinders after the hyphae grow over the fungus cylinders for 7-10 days;

i. opening one end of the cultured fungus cylinder on a superclean bench, transferring the strain into a sterile breathable paper bag, pressing grains with the fungus to disperse, and hanging and airing the grains in a cool and ventilated place;

j. the completely dried strain can be put into a sterile self-sealing plastic bag, a strain label is attached, the name and the date of the strain are noted, and the strain is stored in a dark place at the temperature of 4 ℃; or packaging in vacuum packaging bag, vacuumizing, and storing at-20 deg.C for a long time.

2. The use of the wheat scab grain seed produced by the method of claim 1, comprising the steps of:

firstly, detecting activity: laying a piece of filter paper in a clean culture dish, adding a small amount of sterilized distilled water to completely wet the filter paper, putting 50 grains of strains in each culture dish, completely dispersing the strains, covering the strains, sticking strain labels, placing the strains in a completely dark culture box for constant temperature culture at 25 ℃, observing the growth condition of the strains after 36 hours, indicating that the strains have activity if white strains grow all around the grains of the strains, indicating that the strains lose activity if the grains of the strains do not grow the strains, arranging for 2-3 times of repetition, recording and counting the proportion of the strains with the activity, and if the strains with the activity account for more than 90 percent, using the strains for field inoculation;

inoculating by adopting a single-flower inoculation method, wherein the inoculation period is from after ear emergence of the wheat to before flowering, the inoculation part is the right-side floret of the 6 th floret with the lowest side of the wheat base part and the upward side of the floret, selecting ears with 2-3 days of ear emergence, clamping a grain strain by using a forceps, placing the grain strain into the inner shell of the right-side floret of the target floret, spraying 2-3 times of purified water by using a handheld spraying kettle aiming at the ear of the bacteria, keeping the ears moist, wrapping the ears with a layer of transparent preservative film, or covering the ears with a PE plastic bag for moisturizing, hanging a label, and recording the inoculation time, wherein at least 10 ears are inoculated on each row of materials;

observing the inoculated part after inoculation, preserving moisture for 3-5 days, and taking down a preservative film or a plastic bag after the palea of the inoculated spikelet turns brown;

fourthly, a mist moisturizing system is additionally arranged, water is sprayed for 5-10 times a day, so that the growth environment keeps the humidity more than 50%, the higher the humidity is, the more the gibberellic fungus is expanded, and if the climate is dry, the water spraying times are properly increased;

and fifthly, investigating the scab incidence at 21 days and 28 days after inoculation.