CN106566776A - Space-flight mutation mutant strain Pc-m-6 of pochonia chlamydosporia as well as biocontrol preparation of space-flight mutation mutant strain Pc-m-6 and applications of space-flight mutation mutant strain Pc-m-6 and biocontrol preparation - Google Patents

Abstract

The invention discloses Pc-m-6, a Pc-m-6 mutant strain of P. chlamydoides induced by spaceflight, as well as its biocontrol preparation and application. In the present invention, P. chlamydoides is carried on the Shenzhou 8 spacecraft for space-flight mutagenesis, and the uncarried original strain is used as a control, and the space-flight mutagenesis strain of P. chlamydoides obtained through preliminary screening is carried out including colony growth. Speed, dry weight of mycelia, sporulation and pathogenicity were tested and measured for salt tolerance and resistance to benomyl, and finally a strain with excellent performance and suitable for The mutant strain Pc-m-6 for the biological control of root-knot nematode has high spore production, high pathogenicity to root-knot nematode eggs and excellent resistance to benomyl. The present invention further provides a biocontrol agent prepared by using the aerospace mutagenic strain and its application in controlling root-knot nematode.

Description

Pc-m-6 mutant strain Pc-m-6 of P. chlamydoides and its biocontrol preparation and application

technical field

The present invention relates to space-flight mutation mutant strain of Pochonia chamydosporia=Verticillium chlamydosporium, in particular to the space-flight mutation mutant strain of Pochonia chamydosporia=Verticillium chlamydosporium Pc-m-6 and the biocontrol agent prepared therefrom. The present invention further relates to the application of the mutant strain and the biocontrol preparation prepared from the mutant strain in the control of root-knot nematode, and belongs to the field of screening and application of the mutant strain of P. chlamydoides.

Background technique

Pochonia chamydosporia=Verticillium chlamydosporium belongs to Deuterom ycotina Hyphomycetes and is an important nematode-eating fungus (Lin Maosong, Shen Suwen. Verticillium chlamydosporium) Preliminary report on the control of root-knot nematode incognita [J]. Biological Control Bulletin, 1994, 10(1):7-10) can effectively control root-knot nematode (Meloidogyne incognita), peanut root-knot nematode (Meloidogyne incognita), soybean spore Plant-parasitic nematodes such as Heterodera glycines (Wang Laifa, Yang Baojun, Guan Wengang, etc. Paecilomyces lilacinus and Verticillium chlamydia control root-knot nematode incognita[J], Journal of Sichuan Agricultural University, 1998, 16(2): 231-233; Liu Chang. The parasitic and control effects of Verticillium chlamydia V10 strain on root-knot nematode incognita. Guangxi Agricultural Sciences, 2004,35(2):135-137.). Proconia chlamydoides mainly parasitizes eggs and females through endoparasitism, and kills nematodes through mass reproduction. Control plays an important role and is one of the biocontrol fungi with the most potential development value (Qiu Weifan, Gao Renheng, Liu Xingzhong. Introduction to the biological control of root-knot nematodes [J]. Journal of Guizhou Agricultural College, 1996,15(2):51 -55.), Proconia chlamydoides has wide adaptability, is easy to cultivate, and can parasitize a variety of plant nematodes, so it is beneficial to the application of field control. At the same time, it has been popularized and applied in some countries as a nematode biocontrol agent instead of chemical pesticides, and some achievements have been made. However, due to the complexity of the soil ecosystem, especially the antibacterial effect of soil, the application of biocontrol bacteria is limited.

Space mutation breeding technology, as an effective new technology of mutation breeding, has shown an important role in creating specific mutant gene resources and cultivating new varieties of crops. It has been reported that the Beauveria bassiana isolated from the larvae of the Chinese Shenzhou VIII spacecraft carried Beauveria bassiana, and a mutagenic strain with fast insecticidal speed and high pathogenicity was screened out to control the monochamus alternatus in the forest. It has great application potential (Wang Xizhuo, Wang Laifa, Ma Jianwei, Guo Minwei, Liu Hongjian, Dong Guangping. Screening of highly virulent spaceflight mutagenized strains of Beauveria bassiana [J]. Acta Entomology, 2014, (11 ): 1299-1305.); At the same time, after the Shenzhou 8 spacecraft carried the plant nematode biocontrol bacteria Paecilomyces lilacinus Shandong strain, the biological characteristics of the mutagenized strain were also differentiated to different degrees from the original strain, and the Excellent strains with large positive mutations in growth characteristics and pathogenicity (Wang Yuan, Wang Laifa, Wang Xizhuo, Zhu Tianhui. Biological effects of Paecilomyces lilacinus carried by spaceflight[J]. Nuclear Agricultural Sciences, 2014, (11):1933-1940.). This shows that space-flight mutagenesis has a high mutation rate, abundant mutation types, and increased beneficial mutations, providing more opportunities for breeding excellent strains and providing a new way for breeding excellent strains that meet production needs and have strong stress resistance . Space mutation breeding technology is an effective new technology of mutation breeding. Like other mutation breeding, various traits must be tested after space mutation treatment to judge the effect of space mutation and screen for specific mutation targets. Varieties (Nong Xiangqun, Zhang Zehua, Hu Pan, Gao Song, Zhang Lisheng. Biological Effects of Aerospace Mutagenesis on Entomopathogenic Fungi [J]. Mycology Journal, 2006,25(4):674-681.).

As a biopesticide, P. chlamydoides, like other plant nematode biocontrol bacteria, has many advantages that chemical pesticides cannot match, but at the same time, it also has slow growth, is greatly affected by the environment, unstable control effect and self-stress resistance. Therefore, it is necessary to carry out mutagenesis on the nematode biocontrol fungus P. chlamydoides, and screen out mutant strains with high spore production, strong nematicidal ability, and good resistance to pesticides, so that they can Better play the role of preventing and controlling plant nematodes.

Contents of the invention

The main purpose of the present invention is to carry out aerospace mutagenesis to Pochonia chamydosporia (Pochonia chamydosporia) in order to screen and obtain a strain with high spore production, high pathogenicity to root-knot nematode eggs and relatively effective to benomyl. Mutants with strong resistance and excellent performance;

Another object of the present invention is to apply the obtained mutant strain with excellent performance to biological control of root-knot nematode.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

In the present invention, the cultured and activated P. chlamydobacterium is carried out in spaceflight (Shenzhou No. 8); after the spaceflight mutagenesis is completed, the original bacterial strain of P. chlamydoides that is not carried is used as a control to compare the spaceflight mutagenesis strains. Variability in colony morphology, pigment change, spore morphology, growth rate, spore production, pathogenicity, salt tolerance and benomyl resistance; After the mutagenesis, a wealth of trait variations were produced. Generally speaking, the spaceflight mutation showed different variations in many traits such as growth rate, spore production, pathogenicity, salt tolerance, and benomyl resistance. trend and magnitude. The growth rate, spore production and pathogenicity of strains are the most important indicators for screening biological control strains. Aerospace mutagenesis makes these characteristics show different variation tendencies and ranges, which proves that space mutagenesis is non-fixed, extensive, and positive. Negative bidirectionality, with many mutagenic sites and high mutagenesis rate, the resulting organism variation may have physiological variation or genetic variation (Nong Xiangqun, Zhang Zehua, Hu Pan, Gao Song, Zhang Lisheng. Aerospace mutagenesis Biological effects on entomopathogenic fungi [J]. Mycophyta Sinica, 2006, 25(4): 674~681).

In order to obtain mutants suitable for biological control of nematodes that have high sporulation yield, high pathogenicity to root-knot nematode eggs, and strong resistance to benomyl, the present invention selects numerous chlamydia that are screened. The shape, pigment, mycelium and spore morphology, colony growth rate, mycelia dry weight, sporulation and pathogenicity of the mutant strains of P. Salt tolerance and resistance to benomyl:

The strains subjected to space-flight mutagenesis have different tendencies and magnitudes of variation in sporulation. Among them, the sporulation of the Pc-m-6 mutant strain has a very significant increase compared with the original strain Pc (P<0.05) .

The pathogenicity of P. chlamydoides strains induced by spaceflight mutagenesis to M. incognita had significant variation. Compared with the original strain Pc, 20 strains had an increased parasitic rate on M. incognita eggs. strains, accounting for 69.0% of the mutant strains; 9 strains with reduced parasitic rate, accounting for 31.0% of the mutant strains; among them, the strain with the highest parasitic rate was Pc-m-6, and the parasitic rate reached 94.67%; analysis of variance showed that, The parasitic rate of Pc-m-6 on root-knot nematode eggs was significantly different from that of the original strain (P<0.05), and its higher parasitic rate was very important for field biocontrol practice.

In practical application, the control of root-knot nematode has always been dominated by chemical agents, but the application of chemical agents seriously affects the growth and reproduction of soil microorganisms, and even endangers beneficial microorganisms (Kong Fanyu, Wang Jing. Research progress on root-knot nematode in tobacco plants[J]. Journal of Shenyang Agricultural University, 2001,32(3):232-235.). Therefore, improving the drug resistance of biocontrol bacteria while improving the control effect of biocontrol bacteria can be effectively used in disease control (Zhu Mingliang, Zhang Keqin, Li Feitian, etc. Research progress in biological control of tobacco root-knot nematode[J]. Microbiology Bulletin, 2004, 31(6): 95-99.). The mutant strains induced by spaceflight mutagenesis were screened for benomyl resistance, and the screening results showed that the mutant strains had different variations in resistance to benomyl, among which, the mutant Pc-m-6 showed Very excellent resistance, the difference between its resistance to benomyl (30μg/mL) and the original strain Pc is significant (P<0.05), and the mutant Pc-m-6 can be treated with this concentration of benomyl Stable growth shows that the strain has good compatibility with commonly used fungicides such as benomyl, which will be very beneficial to the popularization and application of P. chlamydoides in production.

Finally, the present invention screens out a mutant strain Pc-m-6 suitable for biological control with excellent performance from numerous aerospace mutant strains. High ability has good resistance to benomyl, the mutant strain grows rapidly, has high spore production, not only has high nematicide activity, but also has good resistance to benomyl, and the control effect is stable, so , the mutant strain Pc-m-6 has important application value as a biocontrol agent in the control of root-knot nematode.

In the present invention, the mutant strain Pc-m-6 of Pochonia chamydosporia (Pochonia chamydosporia) is submitted to a patent-approved institution for preservation, and its microorganism preservation number is: CGMCC No.12510; Pochonia chamydosporia; Preservation time: June 14, 2016; Preservation unit: General Microbiology Center of China Microbiological Culture Collection Management Committee; Preservation address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Academy of Sciences.

The present invention further provides a biocontrol preparation for controlling root-knot nematode, comprising: spore powder or fermented liquid of Pochonia chamydosporia mutant strain Pc-m-6 and a carrier in an effective amount for controlling root-knot nematode.

As a reference, those skilled in the art can refer to the following method to prepare Pochonia chamydosporia (Pochonia chamydosporia) mutant strain Pc-m-6 spore powder:

(1) preparing the Pc-m-6 fermentation liquid of the P. chlamydota mutant strain; (2) reclaiming the spore product from the Pc-m-6 fermentation liquid of the P. chlamydosia mutant strain; (3) drying The recovered Pc-m-6 fermentation product of the P. chlamydoides mutant strain is the spore powder.

Those skilled in the art can prepare the P. chlamydobacterium fermentation broth according to various methods disclosed in the literature; for example, adopt a three-level culture method, that is: seed culture, secondary liquid expansion culture and liquid fermentation culture, Prepare the Pc-m-6 fermented liquid of P. chlamydosia aerospace mutant strain; Wherein, during liquid fermentation culture, can adopt fermentor to ferment or adopt the mode of shake flask culture to carry out fermentation culture; Tertiary culture used The medium and its culture conditions have been disclosed in the literature, as a reference, wherein the composition of the liquid fermentation medium includes a carbon source and a nitrogen source; the carbon source includes but is not limited to glucose, sucrose, maltose, Any one or more of soluble starch or corn flour. The nitrogen source includes but not limited to any one or more of sodium nitrate, ammonium sulfate, peptone or soybean flour.

The present invention further provides a method for preparing a P. chlamydoides biocontrol agent for preventing and controlling root-knot nematodes. The method comprises the following steps: making P. The spore powder or fermented liquid is mixed with the carrier, stirred evenly, pulverized and sieved to prepare the corresponding microbial preparation, for example, it can be granular preparation, powder, wettable powder or microcapsule bacterial preparation.

Wherein, the carrier can be diatomite, kaolin, wood chips, activated carbon, peat, crop straw, dry farmyard manure, etc.; in addition, auxiliary materials or/or And auxiliary agent; described auxiliary material can be crab shell powder or chitin etc.; described auxiliary agent can be wetting agent, dispersant or stabilizing agent etc.

Description of drawings

Fig. 1 is the colony front, back and side morphological types of spaceflight mutagenized Proconia chlamydobacterium;

Fig. 2 is the spore morphology of space-flight mutagenesis P. chlamydobacterium;

Fig. 3 is the histogram of the colony growth rate of spaceflight mutagenesis P. chlamydobacterium;

Fig. 4 is the histogram of spaceflight mutagenesis P. chlamydobacterium strain mycelia dry weight;

Fig. 5 is a histogram of the sporulation yield of P. chlamydus induced by space-flight mutagenesis.

detailed description

The implementation of the present invention will be described in more detail through the following examples, but it should be understood that the examples are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

The preparation of embodiment P. chlamydobacterium wettable powder

One, the preparation of P. chlamydoides spore powder

(1), preparation of fermentation broth

1. After activating the spaceflight mutagenesis mutant strain of P. chlamydoides, carry out shake flask seed culture;

The composition of shake flask seed culture medium (by mass percentage):

Liquid fermentation conditions: put 200ml of seed medium into a 500ml triangular flask, inoculate the spore suspension of Proconia chlamydoides after autoclaving, the inoculation amount is 1.5-3%, place at 24-28°C, shaker 150- 200r/min, cultivate for 18-36h.

2. Secondary liquid expansion culture (by mass percentage):

Medium composition for secondary liquid expansion culture:

The above-mentioned secondary liquid expansion medium is sterilized in place in a fermenter;

Liquid fermentation conditions: fermentation temperature 25-28°C, tank pressure 0.6-0.8bar, oxygen flow rate 100-300L/h, rotation speed 150-250rpm, initial pH value 5.5-6.0.

3. Liquid fermentation culture:

Medium composition of liquid fermentation culture:

Sterilizing the above-mentioned liquid culture medium in-situ in a fermenter;

Liquid fermentation conditions: the fermentation temperature is 28-30°C, the oxygen flow rate can be 100-300L/h, the first 24 hours, the ventilation rate is controlled at 100-150L/h, and the latter is controlled at 250-300L/h; initial pH: 5.0-6.0; rotating speed: 150-200r/min; tank pressure: 0.6-0.8Mpa, the fermentation cycle is controlled at 6 days, and the general microscopic examination spore concentration can reach 10 ⁹ /ml or more.

(2), reclaim and dry spore product from fermented liquid

Centrifuge the fermentation broth under the following conditions: the relative centrifugal force is 4000g, the centrifugation time is 40min, and 2.0g of flocculant is added to every 100ml of fermentation broth; the supernatant is discarded, and the precipitated bacteria slurry is absorbed by diatomaceous earth carrier and dried in the shade to the product If the water content is lower than 10%, the P. chlamydoides spore powder is obtained.

2. Preparation of wettable powder

Weigh each component according to the following mass percentage distribution ratio: 85% of the spore powder prepared above, 11% of diatomite, 2% of wetting agent PEG, and 2% of dispersant wood sodium; mix the spore powder and diatomaceous earth evenly Finally, crush and pass through a 325-mesh sieve; mix the crushed product with a wetting agent and a dispersant evenly, grind it finely with a jet mill, and mix evenly to obtain the product.

Test Example 1 Screening of Pleukonia chlamydobacterium Airborne Mutation Mutant Pc-m-6 and Determination of Mutagenic Effect

1 Materials and methods

1.1 Biomaterials

The tested strain P.chamydosporia originated from the United States and is a production strain used to control root-knot nematodes. The strain is preserved in the Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry Sciences. After spaceflight mutagenesis, it was stored at 4°C in the inventor's laboratory for future use. The root-knot nematode (M.incognita) for the test was provided by the Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry.

1.2 Aerospace carrying

Transfer the hyphae pieces activated by PDA culture for 7 days to 2 sterile EP tubes (1.5 mL) and seal them. One of the tubes was carried on spaceflight (Shenzhou 8), and the other tube was stored in a 4°C refrigerator as a control material.

1.3 Mutant Screening

The spore suspensions were prepared from the P. chlamydota chlamydota and the original strain after the aerospace mutagenesis treatment, and the spore concentration was diluted to 1.0×10 ³ cfu/mL, and spread evenly on a PDA plate with a diameter of 9 cm (per Add 0.1mL spore suspension to the dish), and culture at 25°C for 6d-9d, observe the size, shape and color of the front and back of the single colony, and select the single colony strain with obvious difference from the original strain; the original number is "Pc", and the rest of the strains are The number is "Pc-m-No."

1.4 Detection of aerospace mutagenic effects

Using the uncarried original strain of Proconia chlamydoides as a control, compare the colony shape, pigment change, spore shape, growth rate, sporulation, pathogenicity, salt tolerance and benomyl resistance of the aerospace mutagenized strains. Variability in sex, etc.

1.4.1 Observation of colony morphology and pigment changes

From the grown PDA plate, use a puncher with a diameter of 5 mm to cut 3 colonies into 5 mL of a solution containing 0.5‰ Tween, and shake fully to make a spore suspension. 2.5 μL of spore suspension (spore concentration: 1×10 ⁶ cfu/mL) was placed in the center of a 9 cm petri dish (15 mL medium), cultured in a 25°C incubator, and each strain was repeated 3 times.

1.4.2 Observation of spore morphology

The spore suspension (spore concentration: 1×10 ⁶ cfu/mL) was dropped on a glass slide to make a temporary slide, and the morphology of the spores was observed under a microscope.

1.4.3 Determination of colony growth rate

Dip a sterilized filter paper piece with a diameter of 5 mm and fill it with spore suspension (spore concentration is 1×10 ⁶ cfu/mL), place it in the center of the PDA medium plate, and culture it in a 25°C incubator. Measure the colony diameter on 3d, and stop the measurement until the strain covers the whole dish. The original strain was used as the control and repeated 3 times.

1.4.4 Determination of mycelium dry weight

150 mL Erlenmeyer flasks were filled with 50 mL of PD medium, and 200 μL (spore concentration of 1×10 ⁶ cfu/mL) spore suspension was pipetted into each bottle of culture medium. After culturing for 96 hours, pour all the bacterial liquid in the Erlenmeyer flask into a 50mL centrifuge tube, centrifuge at 5000r/min for 3min, pour off the supernatant, pour the precipitate onto filter paper, dry in an oven at 50°C until constant weight, and weigh.

1.4.5 Determination of spore production

Use a puncher to punch 3 pieces of bacterial blocks with a diameter of 5mm at the midpoint from the center of the colony to the edge, put them into 5mL of a solution containing 0.5‰ Tween, shake fully, and measure the spores with a hemocytometer (25×16 grid) content, repeated 3 times.

1.4.6 Determination of pathogenicity

Collect the diseased roots in the greenhouse, wash the diseased roots, cut them into 0.5cm small pieces, put them into a 500mL triangular flask, pour 200mL of 1% sodium hypochlorite solution, seal it and shake it violently for 3 minutes, quickly pass through a 200-mesh sieve, and then quickly pass through a 500-mesh sieve. The eggs left on the 500-mesh sieve were washed repeatedly with distilled water, and finally washed with sterile water and collected in a small sterile beaker, observed under a microscope, and the concentration of eggs was adjusted to 1000/mL. Add 100 μL of egg suspension to a petri dish with a diameter of 6 cm, and add 2 mL of 1.0×10 ⁶ /mL mutant spore suspension, and wild-type Pc as a control. Each mutant strain was repeated 3 times, cultured at 25°C for 5 days, the number of parasitized eggs and the number of unparasitized eggs were counted, and the parasitic rate of the spores of the strain to the eggs of Meloidogyne incognita was calculated.

1.4.7 Determination of salt tolerance

Prepare PDA medium containing NaCl respectively 0.05mol/L, 0.10mol/L, 0.15mol/L, 0.20mol/L, 0.25mol/L and 0.50mol/L. Dip a filter paper sheet with a diameter of 5 mm of sterilized bacteria and fill it with the spore suspension of the aerospace mutagenic strain (spore concentration is 1×10 ⁶ cfu/mL), put it in the center of the PDA medium containing NaCl at different concentrations above, and place it at 25°C Cultivate in an incubator, measure the colony diameter after 9 days, and calculate the growth rate.

1.4.8 Benomyl resistance screening

Prepare a PDA medium with a final concentration of 30 μg/mL benomyl (wettable powder with 50% benomyl specification, product of Jiangsu Lanfeng Biochemical Co., Ltd.). Dip a sterilized filter paper piece with a diameter of 5mm and fill it with the spore suspension of the aerospace mutagenic strain (spore concentration is 1×10 ⁶ cfu/mL) and place it in the center of the PDA medium with a final concentration of 30 μg/mL benomyl. Cultivate in a 25°C incubator, measure the diameter of the colony after 9 days, and calculate the growth rate.

1.4.9 Data processing and analysis

Data were processed using Microsoft Excel software. SPSS 19.0 was used for one-way analysis of variance (One-way ANOPC-M-A), and the Duncan method was used for multiple comparisons of different bacterial strains (P=0.05). Form representation.

2 Results and Analysis

2.1 Effect of spaceflight mutagenesis on colony morphology and pigment changes

After space-flight mutagenesis of P. chlamydoides obtained stable traits through 3-4 generations of subculture, it was observed that the colonies had obvious morphological differentiation (Fig. 1). The differentiated strains are divided into four types: I, II, III, and IV: the colony shape of type I is similar to that of the original strain Pc, the front of the colony is white and downy, the back of the colony is light yellow, the side of the colony is smooth and arched, and the colony of type I is There were 11 strains in total, accounting for 37.9% of the space-induced mutagenic strains; the colony of Type II strains was white and down-like in front, the center of the colony was sunken and wrinkled, and the color of the back was light yellow, darker than that of Type I. There were 14 strains of Type II colonies, accounting for 48.3% of the mutant strains; type III colonies were white down-like in the front, with no folds in the center of the colony, and light yellow in the back. m-54, accounting for 10.3% of the strains mutated by spaceflight; Type IV colonies are white and downy in front, with white bulges and folds in the center of the colony, and yellow on the back. There is only one strain Pc-m-9 in Type IV colonies, accounting for 3.4% of spaceflight mutagenized strains.

2.2 Effect of spaceflight mutagenesis on spore morphology

Observed under a microscope (100 μm), the shape of the conidia of P. chlamydoides induced by spaceflight is spherical or nearly spherical, transparent and smooth, and the four types (I, II, III, IV) are not significantly different from the original strain Pc. difference.

2.3 The effect of spaceflight mutagenesis on the growth rate of bacterial colonies

According to observations, the growth rate of spaceflight-induced P. chlamydoides is about 1cm/3d, but the growth rate slows down after 15 days, and it takes about 20 days for the colony to cover the entire dish. There were 10 strains whose colony diameter was greater than or equal to the original strain Pc after culturing for 15 days, accounting for 34.5% of the mutant strains; 19 strains were smaller than the original strain Pc, accounting for 65.5% of the mutant strains. Analysis of variance showed that there was no significant difference between the growth rate of the mutant strain Pc-m-6 and the original strain Pc, and the growth rate of the mutant strain Pc-m-38 was faster in the first 9 days, which was significantly different from the original strain Pc (P <0.05), and then the growth rate gradually slowed down (Table 1, Figure 3).

Table 1 The colony growth rate of space-flight mutagenized Proconia chlamydobacterium

Table 1Varieties of colony growth rate of aerospace mutant strains of Pochonia chamydosporia/cm

Note: The data in the table are the mean ± standard deviation, and * after the data indicates that there is a significant difference compared with the original strain Pc (P<0.05). Note: Data in the table is mean±SD, data marked*represents significant difference(P<0.05).

2.4 The effect of spaceflight mutagenesis on the dry weight of mycelia

The dry weight of mycelia showed positive and negative two-way variation during the cultivation of spaceflight mutagenized strains (Figure 4). Among them, Pc-m-26 had the heaviest mycelium dry weight, which was 0.2776g; the mutant strain with the smallest dry weight was Pc-m-27, which had a dry weight of 0.1559g. Analysis of variance showed that the mycelium dry weight of Pc-m-4, Pc-m-27, Pc-m-51 and Pc-m-130 were significantly different from the original strain (P<0.05), including Pc-m- The mycelial dry weight of other mutant strains including 6 was not significantly different from the original strain (P>0.05).

2.5 The effect of spaceflight mutagenesis on sporulation

The test results are shown in Table 2 and Figure 5. The strains after spaceflight mutagenesis showed variations in different tendencies and magnitudes (Table 2 and Figure 5). Compared with the original strain, the sporulation yield of Pc-m-6 has been significantly improved. Analysis of variance showed that there was a significant difference between the sporulation yield of Pc-m-6 and the original strain (P<0.05).

Table 2 The amount of spore production of Proconia chlamydota induced by spaceflight

Table 2 Sporulation of aerospace mutant strains of Pochonia chamydosporia/(×10 ⁶ cfu·mL ^-1 )

Note: The data in the table are the mean ± standard deviation, and * after the data indicates that there is a significant difference compared with the original strain Pc (P<0.05). Note: Data in the table is mean±SD, data marked*represents significant difference(P<0.05).

2.6 The impact of aerospace mutagenesis on pathogenicity

The test results are shown in Table 3. From the test results, it can be seen that the pathogenicity of P. chlamydosia spaceflight mutagenized strains to root-knot nematode incognita has obvious variation. Compared with the original strain Pc, there were 20 strains with increased parasitism rate to M. incognita eggs, accounting for 69.0% of the mutant strains; 9 strains with reduced parasitism rate, accounting for 31.0% of the mutant strains. Among them, the strain with the highest parasitic rate was Pc-m-6, with a parasitic rate of 94.67%; the strain with the lowest parasitic rate was Pc-m-50, with a parasitic rate of 65.33%. The highest positive variation rate of parasitic rate is 12.67%, the highest negative variation rate is -15.67%, and the variation range is between -15.67% and 12.67%. Analysis of variance showed that Pc-m-4, Pc-m-6, Pc-m-10, Pc-m-15, Pc-m-26, Pc-m-38, Pc-m-44, Pc-m- 45. The parasitic rate of Pc-m-50, Pc-m-51, Pc-m-52, Pc-m-123 and Pc-m-130 was significantly different from that of the original strain (P< 0.05), the other strains were not significantly different from the original strain (P>0.05).

Table 3 The parasitic rate of P. chlamydoides induced by spaceflight mutagenesis on the eggs of M. incognita

Table 3 Parasitic rate of aerospace mutant strains of Pochoniachamydosporia against Meloidogyne incognitaeggs/%

Note: The data in the table are the mean ± standard deviation, and * after the data indicates that there is a significant difference compared with the original strain Pc (P<0.05). Note: Data in the table is mean±SD, data marked*represents significant difference(P<0.05).

2.7 The effect of spaceflight mutagenesis on the salt tolerance of strains

The test results are shown in Table 4. The strains subjected to space-flight mutagenesis showed different types of variation in salt tolerance. The first type is that the growth rate accelerates with the increase of the salt concentration, and after reaching a certain value, the growth rate slows down with the increase of the salt concentration, and the optimum growth salt concentration is 0.10-0.15mol/L. There were 17 strains in total, accounting for 58.6% of the mutant strains, and Pc-m-6 and the original strain Pc also belonged to this type. The second type is that the growth rate slows down with the increase of salt concentration; there are 9 mutant strains in this type, accounting for 31.0% of the mutant strains. In addition, there were 3 mutant strains that did not belong to the above two types, namely Pc-m-30, Pc-m-39 and Pc-m-51, accounting for 10.3% of the mutant strains. Analysis of variance showed that the tolerance of Pc-m-6 to high-salt environment (0.50mol/L) was significantly different from that of the original strain Pc (P<0.05), and it could grow stably in high-salt environment.

Table 4 Determination of Salt Tolerance of Aerospace Mutagenesis P. Chlamydobacterium

Table 4Salt tolerance of aerospace mutant strains of Pochonia chamydosporia/cm

Note: The data in the table are the mean ± standard deviation, and * after the data indicates that there is a significant difference compared with the original strain Pc (P<0.05). Note: Data in the table is mean±SD, data marked*represents significant difference(P<0.05).

2.8 The effect of spaceflight mutagenesis on benomyl resistance

The test results are shown in Table 5. After space-flight mutagenesis, different variations of resistance to benomyl appeared in the strains (Table 5). The final concentration of benomyl in the adjustment medium was 30 μg/mL, and the results were observed after culturing at 25°C for 9 days. It was found that the mutant Pc-m-6 showed very strong resistance to benomyl. Analysis of variance showed that Pc-m-4, Pc-m-6, Pc-m-9, Pc-m-10, Pc-m-13, Pc-m-26, Pc-m-27, Pc-m- 30. The resistance of Pc-m-37 and Pc-m-133 to benomyl (30μg/mL) is significantly different from that of the original strain Pc (P<0.05), and they can be stable at this concentration of benomyl grow.

Table 5 Screening of benomyl resistance of spaceflight-induced P. chlamydobacterium

Table 5Benomyl resistance of aerospace mutant strains of Pochoniachamydosporia/cm

Note: The data in the table are the mean ± standard deviation, and * after the data indicates that there is a significant difference compared with the original strain Pc (P<0.05). Note: Data in the table is mean±SD, data marked*represents significant difference(P<0.05).

Experimental example 1 Field experiment of controlling root-knot nematode of pepper with wettable powder prepared by spaceflight mutagenesis mutant strain Pc-m-6 of P. chlamydoides

1 Test materials and methods

1.1 Bacteria to be tested

Test bacterial agent: wettable powder (prepared in Example 1) prepared with P.

Control bacterial agent: wettable powder (prepared according to the method of Example 1) prepared with the original strain of Proconia chlamydota (without aerospace mutagenesis).

1.2 Experimental crops

Pepper trees grown in pepper growing areas.

1.3 Experimental design

The experimental site was set in a pepper planting area with a large and uniform nematode population, and the population of the second instar larvae of the root-knot nematode was 20-30 per 100g of soil in the pre-medication survey.

The experiment is divided into 3 groups, test 1 group, test 2 group and clean water control group. The specific experimental design is as follows:

Test 1 group adopts the wettable powder prepared by embodiment 1 to process;

The control group 2 was treated with the wettable powder prepared in the comparative example.

Clear water control group: use clear water to process (do not apply any nematode control medicament), and do not treat nematodes;

Each treatment has 30 repetitions (divided into three columns, each column has 10 Zanthoxylum bungeanum plants), and each treatment is randomly arranged.

From April 15th to May 10th, carry out the bacterial agent district test, scrape away the soil layer with a thickness of 10 cm on the root surface and a radius of 50 cm around the Zanthoxylum bungeanum, and apply 1 milliliter of the original bacterial agent (the amount of the test bacterial agent and the contrast bacterial agent) All are the same, all are 1 milliliter of original bacterial agent/strain) After diluting with water, adopt spray mode to evenly spray the diluted bacterial agent in the well-dug pit, make the bacterial agent distribute in the root circle, and then reset the soil that is scraped open. 90 days after spraying, the root-knot index (disease index) and the number of second-instar larvae in the soil were investigated to evaluate the control effect.

Calculation method of root-knot index (disease index): dig out every processing prickly ash root, investigate according to root-knot nematode grading investigation index, root-knot severity is divided into 0-4 grades, and the grading standard refers to Benjamin et al. (Benjamin D, Grover C BJ. Comparison of compatible and incompatible response of potato to Meloidogyneincognita. Journal of Nematology, 1987, 19:218-221). The root-knot index calculation formula is as follows:

Calculation method of percentage reduction of larvae (relative reduction rate): collect 5 soil samples from the crop root circumference (0-20cm deep) with a soil drill (2cm×H20cm) in each plot, mix well, take 100g and float by centrifugation Separation method (Karssen G.ThePlant Parasitic Nematode Genus Meloidogyne Goldi, (Tylenchida) in Europe [M]. Gent: Drukkeru Modern, 1982: 5-24.) separates the second instar larvae in the soil sample, and heat kills them with 4% Fix in formalin, count under an inverted microscope, and calculate the number of second-instar larvae of root-knot nematode and the percentage reduction of larvae (relative reduction rate) in 100 g of soil samples. Calculated as follows:

The formula for calculating the relative control effect is as follows:

2 Experimental results

The experimental results are shown in Table 6.

Table 6 Experiments on the control effect of P. chlamydota spaceflight mutagenesis mutant strain Pc-m-6 wettable powder on root-knot nematode of pepper

From the test data of table 6, it can be seen that the relative control effect of the test bacterial agent (prepared with P. chlamydobacteria spaceflight mutagenesis mutant strain Pc-m-6) to root-knot nematode is better than that of the control bacterial agent (with The wettable powder prepared by the original bacterial strain of P. chlamydoides) was 23.11 percentage points higher, and the larva reduction percentage of the test bacterial agent was 20.2 percentage points higher than that of the control bacterial agent. The field test results of root-knot nematode control showed that the biological control effect of Pc-m-6 of P. chlamydosia space mutation mutant on root-knot nematode was much better than that of the original strain on root-knot nematode.

Claims (10)

1. one plant thick wall Pu Keniya bacterium (Pochonia chamydosporia) Flight Mutagenesis mutant Pc-m-6, its feature exist In its microbial preservation number is：CGMCC No.12510.

2. the thick wall Pu Keniya bacterium Flight Mutagenesis mutant Pc-m-6 described in claim 1 preventing and treating plant insect in should With.

3. according to the application described in claim 2, it is characterised in that：Described plant insect is nematicide.

4. according to the application described in claim 3, it is characterised in that：Described nematicide is root-knot nematode.

5. it is a kind of preventing and treating plant insect biological prevention and control agent, it is characterised in that include：In preventing and treating described in the claim 1 of effective dose Thick wall Pu Keniya bacterium Flight Mutagenesis mutant Pc-m-6 spore powder or fermentation liquid and carrier.

6. according to the biological prevention and control agent described in claim 5, it is characterised in that：Described carrier be kieselguhr, Kaolin, wood flour, The farm manure of activated carbon, turf, agricultural crop straw or drying.

7. according to the biological prevention and control agent described in claim 5, it is characterised in that：Also contain adjuvant or auxiliary agent.

8. according to the biological prevention and control agent described in claim 7, it is characterised in that：Described adjuvant is crab shell powder or chitin；It is described Auxiliary agent be wetting agent, dispersant or stabilizer.

9. according to the biological prevention and control agent described in claim 5, it is characterised in that：Described plant insect is root-knot nematode.

10. a kind of method for preparing biological prevention and control agent described in claim 5, comprises the following steps：(1) cultivate described in claim 1 Thick wall Pu Keniya bacterium Flight Mutagenesis mutant Pc-m-6, obtain its spore powder or fermentation liquid；(2) by the spore powder for being obtained Or fermentation liquid and carrier are mixed, and are stirred, and are pulverized and sieved, and obtain biological prevention and control agent.