CN112205118A - Method for symbiotic radish plants in beauveria bassiana and defense of symbiotic plants against plutella xylostella - Google Patents

Abstract

The invention relates to a method for symbiotic radish plants in beauveria bassiana and defense of the symbiotic plants against plutella xylostella. The method comprises the following steps: sterilizing radish seed surface, drying with sterile absorbent paper, and pouring the seed into the containerBeauveria bassianaSoaking the seeds in an erlenmeyer flask of the spore suspension; the beauveria bassiana strain is obtained from the China forestry microorganism preservation management center, is separated from the larva of anoplophora glabripennis and is then stored in the Beina strain storage center, and the strain preservation number is BNCC 117565; the colonization method provided by the invention is simple, convenient and effective,B.bassianathe endosymbiotic radish can inhibit the feeding and growth development of the plutella xylostella, improve the death rate of larvae, has positive significance in the aspect of biologically preventing and controlling the plutella xylostella,meanwhile, the process of biological control by using beauveria bassiana as endophytic fungi is promoted.

Description

Method for symbiotic radish plants in beauveria bassiana and defense of symbiotic plants against plutella xylostella

Technical Field

The invention relates to a method for symbiotic radish plants in beauveria bassiana and defense of the symbiotic plants against plutella xylostella.

Background

Endophytic fungi are a class of fungi that are parasitic in healthy plants and do not cause infestation. The endophytic fungi often have favorable influence on symbiotic host plants, can promote the growth and development of the host plants, can enhance the resistance of the host plants to pests, and indirectly inhibit the pests. Entomopathogenic fungi, as pathogenic natural enemies that directly poison pests, are an important biological resource for controlling pests. In recent years, research has been carried out to record that entomopathogenic fungi have plant endophytes and can directly generate nutritional relationship with plants in natural environment or by adopting an artificial inoculation mode, wherein beauveria bassiana is the currently described entomopathogenic fungi which can be colonized in various plants. The beauveria bassiana becomes endophytic fungi which continuously colonizes in plants by artificial inoculation means such as spraying, seed soaking, root irrigation and the like, so that the growth of the plants can be promoted, and the resistance of the plants can be improved. At present, foreign researches on improvement of plant insect resistance by using beauveria bassiana as a plant endophytic fungus are deep, but domestic related researches are few, and no research on beauveria bassiana as a radish endophytic fungus exists at home and abroad.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for symbiotic radish plants in beauveria bassiana and defense of the symbiotic radish plants against diamond back moths, wherein seeds of radish are treated by a beauveria bassiana spore suspension seed soaking method to colonize in radish, and the seed soaking method is found to enable radish seeds to colonizeB. bassianaEffectively colonizes the radish plant.B. bassianaCan inhibit the eating and the growth of the plutella xylostella after the radish is symbiotic, has positive significance in the aspect of biologically preventing and controlling the plutella xylostella, has certain application prospect, is favorable for comprehensively evaluating multiple values of the beauveria bassiana as endophytic fungi of the radish, promotes the process of biologically preventing and controlling the beauveria bassiana as the endophytic fungi, and accordingly generates more remarkable economic benefitSocial and ecological benefits.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for symbiotic radish plants in beauveria bassiana comprises the following steps: sterilizing the surfaces of radish seeds, sucking the water on the surfaces of the seeds by using sterile absorbent paper after the sterilization is finished, and then pouring the seeds into a prepared conical flask filled with beauveria bassiana spore suspension for seed soaking.

The surface disinfection method comprises the following steps: soaking in 75% ethanol solution for 3 min, soaking in 1.5% sodium hypochlorite solution for 3 min, and washing with sterile water for 10 s each time for 3 times.

The concentration of the beauveria bassiana spore suspension is 1 multiplied by 10⁸Per mL; the solvent for the suspension was a 0.05% Tween-80 solution.

The seed soaking conditions are that the temperature is 25 ℃, the rotating speed is 110 r/min, and the seed soaking time is 12 h.

The radish product is named as big radish in southern panzeria.

Further, the radish plant obtained by the method is applied to defense of diamondback moth.

Compared with the prior art, the invention has the following beneficial effects:

provides a new record for the plant endogenous research of beauveria bassiana, and finds that the beauveria bassiana can be cultured by a seed soaking methodB. bassianaEffectively colonize radish plants, andB. bassianathe endosymbiotic radish can inhibit the feeding, growth and development of plutella xylostella.

Drawings

FIG. 1:B. bassianacolonizing radish leaves, stems and roots.

FIG. 2:B. bassianaeffect of colonization in radish on radish plant height.

FIG. 3:B. bassianaeffect of colonization in radish on radish leaf length.

FIG. 4:B. bassianaeffect of colonization in radish on radish leaf width.

FIG. 5:B. bassianacolonization in radish has a high impact on radish stem.

FIG. 6:B. bassianaeffect of colonization in radish on radish root length.

FIG. 7:B. bassianaeffect of colonization in radish on dried radish plant weight.

FIG. 8:B. bassianaeffect of colonization in radish on diamondback moth intake.

FIG. 9:B. bassianathe effect of colonization in radish on the weight of cabbage moth fourth-instar larvae and pupae.

FIG. 10:B. bassianathe effect of colonization in radish on the development history of plutella xylostella; wherein A is the developmental stage (except for the egg stage); b is a larval stage; c is pupal stage; d is the adult stage.

FIG. 11:B. bassianathe effect of colonization in radish on the propagation of plutella xylostella; wherein A is pupation rate; b is eclosion rate; c is the single female egg laying amount; d is the hatching rate of the eggs.

FIG. 12B. bassianaThe effect of colonization in radish on diamondback moth mortality; wherein A is larval mortality; and B is the death rate of pupa.

Detailed Description

The invention is further explained below with reference to the drawings and the embodiments.

Example 1

The embodiment provides a method for symbiotic radish plants in beauveria bassiana and defense of the symbiotic plants against plutella xylostella. The beauveria bassiana strain is obtained from the China forestry microorganism preservation management center, is separated from the larva of anoplophora glabripennis and is then stored in the Beina strain storage center, and the strain preservation number is BNCC 117565.

The diamondback moth was collected in 2004 in a new rural area (26.08 ° N, 119.28 ° E) in fujian province, and was kept for a long time in a laboratory phytotron.

The method comprises the following steps:

the invention adopts potted planting. The specification of the planting pot is 20 x 10 x 8cm, and 100 grains are planted in each potB. bassianaThe treated seeds are 5 pots, and each pot is provided with a separate mesh cage; the control group was planted in the same manner. The experiment was set up for 3 biological replicates. Fruit of Chinese wolfberryThe test treatment comprises the following steps: pouring the seeds into a conical flask filled with beauveria bassiana spore suspension in advance, wherein the concentration of the beauveria bassiana spore suspension is 1 × 10⁸Per mL; the solvent for the suspension was a 0.05% Tween-80 solution. The control group is prepared by soaking radish seeds in sterile water containing 0.05% Tween-80, placing two groups of conical bottles in a shaking table at 25 deg.C and 110 rpm for 12 hr, and taking out. Sterile water is poured for 1 time every day (the time is fixed), the used artificial climate chamber is sterilized by ultraviolet rays for 4 hours in advance, the planting temperature is 25 +/-2 ℃, the humidity is 70 +/-5%, and the photoperiod is 16L to 8D.

And (3) performing surface disinfection on the randomly selected 12 seeds (the time for surface disinfection is 4 min, namely soaking in 75% ethanol and 1.5% sodium hypochlorite solution, finally cleaning for 3 times for 10 s each time by using sterile water), and sucking water on the surfaces of the seeds by using sterile absorbent paper after disinfection is finished. Calculating the number of spores in the suspension by using a blood counting plate, wherein the concentration of the spore suspension is 1 multiplied by 10⁸one/mL, stored at 4 ℃ for later use. The soil is selected from natural peat soil, subpackaged in tin paper boxes, put into a high-pressure steam sterilization pot, set at 121 ℃ for 1 h, dried in a forced air drying oven at 100 ℃ for 48 h, and cooled in a sterile artificial climate chamber before use.

Example 2

Referring to FIGS. 1-12, this example provides a method and results for detecting the effects of example 1.

First, experiment method

1. B. bassianaDetection of colonization in radish

Example 1 after one week, 3 radish seedlings were randomly taken out from each pot, washed with clean water to minimize damage to the plants, each seedling was cut into 2 leaves, 2 stems and 2 roots with a sterile scalpel, the leaves, stems and roots of each seedling were subjected to surface sterilization, respectively, and after surface sterilization was completed, the seedlings were dried in the air (water was absorbed with a sterile paper towel to accelerate drying) and placed on an YPDA plate, cultured in an artificial incubator, and observed after 7 days. Sterile water-treated seeds were tested in the same manner. The assays were performed 1 time per week for 4 weeks, with 3 biological replicates per pot. In 5 basins after four weeksB. bassianaSelecting radish seedlings in a planting pot with high colonization rate to carry out experiments on the feeding amount and the fitness of the plutella xylostella. Colonization rate (%) = colonizationB. bassianaNumber of leaves, stems, roots/total number of leaves, stems, roots.

2. Endogenetic of radish young plantB. bassianaIdentification of

The endophytic fungi in young radish plant can grow to 3-4 cm at 7 days²The strain with the size is white or light yellow in color, and the primary identification isB. bassianaSelecting endogenesis with small amount of inoculating loop or fine bamboo stickB. bassianaThe spores and hyphae were put on a sterile glass slide to prepare a slide, stained with Cotton Blue, and observed under an optical microscope. Endogenous with sterile bladesB. bassiana1 cm of the strain was excised²And extracting DNA from the fungus block with the size for molecular identification. DNAMAN software for sequencing results andB. bassianathe sequences of the mother species were analyzed by alignment.

Inoculation ofB. bassianaWhen the radish grows to 2 nd week, taking radish leaf, sterilizing the surface, slicing with sterile blade, and examining with optical microscopeB. bassianaColonization and growth in radish leaves.

3. Detection of radish seed germination rate and growth and development

And recording the germination condition of the seeds 5 days before sowing, counting the germination rate, and setting 5 biological repetitions in the experiment. The plant height, leaf length, leaf width, stem height, main root length, and dry plant weight of young radish plants were recorded every other week for 4 weeks for comparisonB. bassianaWhether the growth and development of the treated seedlings are different from those of the control group seedlings or not. The specific method comprises the following steps: 3 radish seedlings were randomly picked out from each pot, washed with clear water and damaged as little as possible. The surface water is sucked dry by absorbent paper, and the plant height, leaf length, leaf width, stem height and main root length of the plant are measured by a measuring ruler, wherein the measuring unit is mm. When measuring the length and width of the leaf, measuring two cotyledons of the seedling before the cotyledons are not fallen off, taking an average value, and measuring only true leaves after the cotyledons are yellowed and shrunk; stem height refers to the length above the root and below the petiole; finally, putting the whole seedlings into an oven, setting the temperature at 80 ℃, and weighing the dry weight after 24 hours by using an analytical balance (OHAUS). The two sets of measurements were kept consistent with time, 4 weeks of testing once a week, with 3 biological replicates per pot.

4. B. bassianaEffect of colonization in radish on the amount of diamond back moth eaten

After sowing the seeds, the test was performed every other week from week 2B. bassianaThe effect of colonization in radish on the amount of plutella xylostella consumed was examined for a total of 3 weeks. The 3 rd larvae of FZss diamondback moth are picked about 500 times before detection and starved for 8 h. Select outB. bassianaAnd (3) in planting pots with the colonization rate of the leaves being more than or equal to 60%, randomly selecting 10-15 seedlings in each pot, cleaning the seedlings with sterile water, absorbing water on the surface of the seedlings with absorbent paper, shearing off radish leaves with sterile scissors, placing the radish leaves in an analytical balance, weighing 0.5 g of the radish leaves, accurately measuring the radish leaves to four digits after decimal point, and recording a specific numerical value. Weighing, and putting the radish leaves into a paper box for later use. During sampling, additionally weighing an equal amount of vegetable leaves for calculating dry weight, and putting the vegetable leaves into a drying box for drying at 80 ℃ for 24 h and then weighing. Putting the diamondback moth larvae hungry for 8h into paper boxes with leaves, putting 15 heads in each box, picking out the diamondback moth larvae with forceps after eating for 12 h, washing the eaten leaves with sterile water, putting the leaves into a drying box for drying at 80 ℃, and weighing the dried leaves with an analytical balance after 24 h. The two groups of detection methods are the same. Each basin is provided with three repetitions, and the number of basins is controlled byB. bassianaThe colonization rate of (2). The assay was performed 1 week apart for 3 weeks starting at week 2. Feed intake per head of larvae = (dry weight before feed-dry weight after feed)/total number of surviving plutella xylostella.

5. B. bassianaEffect of colonization in radish on the fitness of Plutella xylostella

Selecting the seeds 2 weeks after sowingB. bassianaPlanting pots with the colonization rate of the leaves being more than or equal to 60 percent are selected, 21 radish seedlings are randomly selected from each pot, the seedlings are cleaned by sterile water, surface water is absorbed by absorbent paper, the seedlings are evenly divided into 3 parts, the roots of the seedlings are wrapped into one bundle by wet cotton in each part, and the bundles are respectively placed into paper boxes filled with filter paper for standby. Picking about 500 heads of 1-year-old plutella xylostella larvae with a soft brush pen, putting the larvae into paper boxes filled with seedlings, and putting 15 heads in each paper box. The seedlings in the paper box are replaced every two days, the growth condition of the diamondback moth is observed and recorded every day, and the indexes to be recorded comprise: the weight of 4 instar insects, the weight of pupae, the eclosion rate, the egg laying amount per female, the hatching rate, the death rate of larvae, the death rate of pupae and the development period. Weighing each 4-year-old larva, recording, independently feeding each diamond back moth in a disposable culture dish with small holes after weighing, and replacing the number of the fed seedlings by 2 seedlings every two days. And recording the pupation time of each diamondback moth, weighing each pupa for recording when the color of the diamondback moth pupae is observed to be yellow-green, and avoiding puncturing pupa clothes when weighing. Weighing, and placing each diamondback moth into a transparent small bottle with diameter of 1.5 cm and height of 1.5 cm, and plugging clean cotton at the bottle mouth. After the plutella xylostella eclosion, recording eclosion time, taking the plutella xylostella eating seedlings in the same pot for pairing 15 pairs, feeding the plutella xylostella by dripping 15% of honey water on cotton at the bottle mouth, and replacing a small bottle with a new one every day. And recording the number of eggs laid by the plutella xylostella in the first three days and counting the hatchability of the eggs on the next day. Growth status of each diamondback moth, including unsuccessful pairings, was recorded until death, and the time of death was recorded.

The experiment was performed for data analysis using statistical software spss.21.0, plotted using GraphPad Prism and Excel 2013 software.

Second, results and analysis

1. B. bassianaColonization in radish plants

No detection in the shoot roots of the control seedlingsB. bassianaThe colonization of (1).B. bassianaThe treated seedlings were grown from the first week to the fourth week,B. bassianathe colonization rates in the leaves were, in order: 33%, 73%, 76%, 79%; the colonization rate in the stem is as follows: 46%, 50%, 67%; the low colonization rates in the roots are in turn: 83%, 100%, 75% (fig. 1). The experimental results show that the product is within four weeksB. bassianaThere is a higher colonization rate in seedlings and the highest colonization rate in roots, followed by leaves and stems.

2. B. bassianaEffect of colonization in radish on the growth of radish plants

B. bassianaThe height of the treated radish plants is one to four sides: (58.40 + -1.98) mm, (73.4 + -3.34) mm, (78.10 + -3.02) mm, and (79.6 + -2.29) mm; sterileThe height of the radish plant subjected to water treatment is one to four sides respectively: (51.3 +/-2.10) mm, (67.8 +/-2.94) mm, (79.30 +/-2.77) mm and (77.2 +/-5.18) mm. The data of each week are analyzed statistically, and the two groups of comparison results in each week are respectively: first week (T = 2.451; P =0.040 < 0.05), second week (T = 1.259; P > 0.05), third week (T = 1.009; P > 0.05), fourth week (T = 0.428; P > 0.05) (FIG. 2). The results show that the plant heights of the two groups of radishes have no significant difference in other weeks except the first week.

And (3) detecting the leaf length, detecting the leaf length of the cotyledon before the cotyledon atrophy turns yellow, and detecting the leaf length of the true leaf after the cotyledon atrophy turns yellow.B. bassianaThe length of the treated radish leaves is one to four weeks as follows: (5.7 +/-0.19) mm, (7.9 +/-0.33) mm, (18.4 +/-1.11) mm and (21.9 +/-0.37) mm; the leaf length of the radish treated by sterile water is one to four weeks as follows: (5.4 +/-0.10) mm, (7.2 +/-0.29) mm, (18.3 +/-1.47) mm and (21.8 +/-0.56) mm. The two groups of comparison results in each week are respectively: first week (T = 1.445; P > 0.05), second week (T = 1.570; P > 0.05), third week (T = 0.084; P > 0.05), fourth week (T = 0.570; P > 0.05) (FIG. 3). The results show that the leaf length of the radishes in the two groups has no significant difference within four weeks.

B. bassianaThe leaf width of the treated radish is one to four: (8.2 +/-0.10) mm, (9.4 +/-0.24) mm, (10.3 +/-0.28) mm and (12.8 +/-0.48) mm; the leaf width of the radish subjected to sterile water treatment is one to four sides respectively: (7.9 +/-0.199) mm, (8.3 +/-0.21) mm, (10.2 +/-0.39) mm and (11.1 +/-0.30) mm. The two groups of comparison results in each week are respectively: first week (T = 1.285; P > 0.05), second week (T = 3.342; P =0.01 < 0.05), third week (T = 0.135; P > 0.05), fourth week (T = 0.150; P > 0.05) (fig. 4). The results showed that the leaf widths of the two groups of radishes were not significantly different except for the second week.

B. bassianaThe stem height of the radish to be treated is one to four weeks as follows: (36.1 +/-1.28) mm, (44.9 +/-2.22) mm, (42.7 +/-1.51) mm and (36.0 +/-2.45) mm; the stem height of the radish subjected to sterile water treatment is one to four weeks as follows: (31.8 + -1.52) mm, (41.3 + -2.39) mm, (41.13 + -1.33)) mm, (36.7 +/-2.11) mm. The two groups of comparison results in each week are respectively: first week (T = 2.183; P > 0.05), second week (T = 1.125; P > 0.05), third week (T = 0.747; P > 0.05), fourth week (T = 0.228; P > 0.05) (fig. 5). The results show that the stem heights of the radishes in the two groups have no significant difference within four weeks.

B. bassianaThe length of the main roots of the radish to be treated is one to four: (6.33 +/-0.69) mm, (4.47 +/-0.65) mm, (6.35 +/-0.50) mm and (5.27 +/-0.37) mm; the main root length of the radish subjected to sterile water treatment is one to four weeks as follows: (4.2. + -. 0.55) mm, (5.5. + -. 0.64) mm, (5.67. + -. 0.45) mm, and (6.10. + -. 0.44) mm (FIG. 6). The two groups of comparison results in each week are respectively: first week (T = 2.409; P < 0.05), second week (T = 1.095; P > 0.05), third week (T = 1.009; P > 0.05), fourth week (T = 1.391; P > 0.05). The results showed that there was no significant difference in root length between the two groups of radishes except for the first week.

B. bassianaThe dry weight of the treated radish for one to four weeks was: (25.38 + -1.09) mg, (24.9 + -0.94) mg, (33.98 + -1.35) mg, (30.88 + -2.36) mg; the dry weight of the radish subjected to sterile water treatment is one to four weeks respectively: (25.32 + -1.06) mg, (24.72 + -1.03) mg, (33.08 + -0.98) mg, and (31 + -2.73) mg. The data of each week are analyzed statistically, and the two groups of comparison results in each week are respectively: first week (T = 0.039; P > 0.05), second week (T = 0.129; P > 0.05), third week (T = 0.540; P > 0.05), fourth week (T = 0.033; P > 0.05) (fig. 7). The results showed that the dry weight of both groups of radishes did not differ significantly within four weeks.

3. B. bassianaEffect of colonization in radish on the fitness of Plutella xylostella

Due to the fact thatB. bassianaThe colonization rate in the radish leaves grown for the first week was low, so that only the feeding amount of plutella xylostella to the radish leaves grown for the second to fourth weeks was examined. Select outB. bassiana4 planting pots with the colonization rate of the leaves being more than or equal to 60 percent are selected, radish seedlings are randomly selected to feed 3-year-old terminal plutella xylostella larvae starved for 8 hours in each pot, and the dry weight of the fed leaves is measured after 12 hours. Calculate the food intakeB. bassianaThe dry weight of the treated radish from two to four weeks was: (2.94 +/-0.10) mg, (3.02 +/-0.11) mg and (2.95 +/-0.11) mg, wherein the dry weight of the radish subjected to sterile water treatment is two to four weeks: (3.25. + -. 0.11) mg, (3.47. + -. 0.10) mg, and (3.34. + -. 0.11) mg. The two groups of comparison results in each week are respectively: statistically significant differences were observed in the second week (T = 4.157; P =0.03 < 0.05), the third week (T = 4.807; P =0.03 < 0.05), the fourth week (T = 4.062; P =0.04 < 0.05) (fig. 8).

Select outB. bassiana4 planting pots with the colonization rate of more than or equal to 60 percent in the leaves are selected respectively to feed the radish seedlings in the 4 pots with the plutella xylostella. The sterilized water-treated radish was selected from 4 pots having the same number. Food takingB. bassianaThe weight of the diamondback moth larvae for processing the radishes is (4.60 +/-0.16) mg, the weight of the diamondback moth larvae for eating the sterilized water-processed radishes is (5.80 +/-0.24) mg, and the two groups have a significant difference (T = 4.288; P =0.03 < 0.05) statistically (FIG. 9); food takingB. bassianaThe pupal weight of the radish-treated diamond back moth was (4.88. + -. 0.21) mg, the pupal weight of the radish-treated diamond back moth eaten with sterile water was (5.10. + -. 0.13) mg, and there was no statistically significant difference between the two groups (T = 0.813; P > 0.05) (FIG. 9). The experimental results show that:B. bassianathe treated cabbage moth larvae have significant influence on the larva weight of the cabbage moth.

Eating with Plutella xylostellaB. bassianaAnalyzing the result of the radish seedling after the treatment and the sterile water treatment from the development period, and taking the radish seedling for eatingB. bassianaThe development duration (except egg phase) of the plutella xylostella of the radish treated by the sterile water is as follows: (23.95. + -. 0.50) d, (23.26. + -. 0.17) d, and no significant difference was found by statistical analysis between the two groups (T = 1.300; P)>0.05) (fig. 10A); food takingB. bassianaThe larva stages of the plutella xylostella of the radish treated by the sterile water are respectively as follows: (9.32 + -0.14) d, (9.27 + -0.17) d, and statistical results showed no significant difference in larval stages between the two groups (T = 0.243; P)>0.05) (fig. 10B); food takingB. bassianaThe pupal stages of the plutella xylostella of the radish treated by the sterile water are as follows: (4.80 +/-0.14) d and (4.71 +/-0.12) d, and the statistical result shows that the pupal stage of the two groups has no difference in significanceIso (T = 0.648; P)>0.05) (fig. 10C); food takingB. bassianaThe adult stages of the plutella xylostella of the radish treated by the sterile water are respectively as follows: (9.8 +/-0.37) d and (9.3 +/-0.26) d, and the statistical result shows that the adult stages of the two groups have no significant difference (T = 1.228; P)>0.05) (fig. 10D). The experimental results show that the food is takenB. bassianaThe colonized radish has no significant influence on the development period of the plutella xylostella.

Eating with Plutella xylostellaB. bassianaAfter the radish seedlings are treated and sterilized by water, the pupation rate, the emergence rate, the single-female egg laying amount and the hatching rate data of the plutella xylostella are analyzed, and the radish seedlings are eatenB. bassianaThe pupation rates of the plutella xylostella of the radish treated by the sterile water are respectively as follows: (90.67. + -. 1.25)%, (89. + -. 1.25)%, and no significant difference was found between the two groups by statistical analysis (T = 0.756; P)>0.05) (fig. 11A); food takingB. bassianaThe emergence rates of the plutella xylostella of the radish treated by the sterile water are respectively as follows: (71.02 + -4.68)%, (73.68 + -1.08)%, and no significant difference was found between the two groups by statistical analysis (T = 1.371; P)>0.05) (fig. 11B); food takingB. bassianaThe single female egg laying amount of the plutella xylostella of the radish treated by the sterile water is respectively as follows: (88.50 + -4.0) and (92.70 + -4.7) grains, and no significant difference was observed between the two data (T = 1.194; P)>0.05) (fig. 11C); food takingB. bassianaThe egg hatchability of the plutella xylostella of the radish treated by the sterile water is respectively as follows: (88.50 + -0.14)%, (88.56 + -0.65)%, and no significant difference was found between the two groups of data by statistical analysis (T = 0.068; P;)>0.05) (fig. 11D). Thus, take foodB. bassianaThe colonized radish has no obvious influence on the pupation rate, the eclosion rate, the single-female egg laying amount and the hatching rate of the plutella xylostella.

Eating with Plutella xylostellaB. bassianaAfter the radish seedlings are treated and sterilized by water, the death rate of the larvae of the plutella xylostella is analyzed, and the radish seedlings are eatenB. bassianaThe larval mortality rates of plutella xylostella treated with radish and sterilized water were: (16.93 ± 1.28)%, 12.67 ± 1.25)%, there was a significant difference in the mortality of the diamondback moth larvae in the two groups (T = 4.072; df = 6; p =0.021 < 0.05) (fig. 12A);B. bassianahyphae grow on the surface of the dead larva of the treatment group, and the sterile water treatment group dies normally. Food takingB. bassianaThe pupa mortality rates of the plutella xylostella of the radish treated by the sterile water and the radish treated by the sterile water are respectively as follows: (18.92 +/-1.55)%, (16.08 +/-1.53)%, and the mortality rates of the Plutella xylostella pupae of the two groups have no significant difference (T = 2.840; P > 0.05) (FIG. 12B).

The results indicate that the endophytic fungi are a class of fungi that parasitize healthy plants and do not cause infestation. The endophytic fungi often have favorable influence on symbiotic host plants, can promote the growth and development of the host plants, can enhance the resistance of the host plants to pests, and indirectly inhibit the pests. Entomopathogenic fungi, as pathogenic natural enemies that directly poison pests, are an important biological resource for controlling pests. In recent years, research has been carried out to record that entomopathogenic fungi have plant endophytes and can directly generate nutritional relationship with plants in natural environment or by adopting an artificial inoculation mode, wherein beauveria bassiana is the currently described entomopathogenic fungi which can be colonized in various plants. The invention utilizes a seed soaking method to soak radish (radish)Raphanus sativusL.) Beauveria bassiana (balsamo) Vuillemin: (L.)B. bassiana) Endophytic fungi, analysisB. bassianaEndophytic fungi for radish growth and phytophagous insect pests diamondback moth: (Plutella xylostellaL.) growth and development, so as to better understand the three-level interaction relationship of radish-beauveria bassiana-plutella xylostella, and is helpful for exploring a new strategy for carrying out comprehensive plant protection biological control application by combining plant endophytic fungi and entomopathogenic fungi preparations.

The invention discovers that the seed soaking method can be used for preparing the seedB. bassianaEffectively colonize in the turnip plant, and do not influence the growth and development of turnip plant to this feeds diamondback moth and can restrain the feeding and growth and development of diamondback moth, and improve the mortality of larva.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (6)

1. A method for symbiotic radish plants in beauveria bassiana is characterized by comprising the following steps: sterilizing the surfaces of radish seeds, sucking the water on the surfaces of the seeds by using sterile absorbent paper after the sterilization is finished, and then pouring the seeds into a prepared conical flask filled with beauveria bassiana spore suspension for seed soaking.

2. The method of claim 1, wherein the surface disinfection method comprises: soaking in 75% ethanol solution for 3 min, soaking in 1.5% sodium hypochlorite solution for 3 min, and washing with sterile water for 10 s each time for 3 times.

3. The method of claim 1, wherein the concentration of the beauveria bassiana spore suspension is 1 x 10⁸Per mL; the solvent for the suspension was a 0.05% Tween-80 solution.

4. The method for producing a symbiotic radish plant with beauveria bassiana according to claim 1, wherein the seed soaking conditions are 25 ℃, 110 rpm and 12 h.

5. The method of claim 1, wherein the radish is named as daikon radish in southern Pacific lake.

6. Use of a radish plant obtained by the method of claim 1 for protection against plutella xylostella.

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