CN106554923B

CN106554923B - Spaceflight mutagenesis strain of chlamydomonas and application thereof

Info

Publication number: CN106554923B
Application number: CN201610885793.9A
Authority: CN
Inventors: 汪来发; 王曦茁; 孟繁丽; 朴春根; 田国忠; 刘洪剑
Original assignee: Research Institute of Forest Ecology Environment and Protection of Chinese Academy of Forestry
Current assignee: Research Institute of Forest Ecology Environment and Protection of Chinese Academy of Forestry
Priority date: 2016-10-10
Filing date: 2016-10-10
Publication date: 2020-01-10
Anticipated expiration: 2036-10-10
Also published as: CN106554923A

Abstract

The invention discloses a chlamydosporia aerospace mutant strain and application thereof. According to the method, chlamydosporia strain is carried on a Shenzhou No. 8 airship for space mutation, an original strain which is not carried is used as a contrast, the chlamydosporia space mutation strain obtained through preliminary screening is subjected to biological detection including colony growth speed, hypha dry weight, sporulation quantity, pathogenicity and the like, the salt tolerance and the benomyl resistance are measured, and finally, the mutant strain Pc-m-130 with good salt tolerance and strong pathogenicity to root-knot nematode eggs is obtained through screening, has strong stress resistance and can stably survive in saline-alkali soil for a long time. The invention further provides a biocontrol agent containing the space mutation strain and application thereof in preventing and controlling root-knot nematodes.

Description

Spaceflight mutagenesis strain of chlamydomonas and application thereof

Technical Field

The invention relates to Pochonia chlamydosporia (Pochonia chamydosporia) space mutation mutant strains, in particular to Pochonia chlamydosporia space mutation mutant strains Pc-m-130 and a biocontrol agent prepared from the Pochonia chlamydosporia space mutation mutant strains, further relates to application of the mutant strains in biological control of root-knot nematodes, and belongs to the field of screening and application of Pochonia chlamydosporia mutant strains.

Background

Pochonia chlamydosporia (Pochonia chamycodospora) belongs to the subdivision Deuterom mycinotina (Hyphomycetes) and is an important plant-feeding nematode fungus (Loranthus chinensis, Proteus meretrix, Proteus fargesii. for the prevention and treatment of Meloidogyne incognita, 1994,10(1):7-10) which is effective in the prevention and treatment of plant parasitic nematodes such as Meloidogyne incognita, Meloidogyne arachidis (Meloidogyne arenaria), Heterocystis glycines (Heterodera globinus) (Meloidogyne arenaria, Meloidogyne guanica, et al. Penicillium 2004 and Verticillium agroticum for the prevention and treatment of Meloidogyne incognita [ J ], Tetrachuan report, 1998,16(2): 231. chrysosporium strain, 10, and Nostoc. chrysospora guanidium, 35). The Pochonia chlamydosporia is mainly parasitized in eggs and female bodies in an internal parasitizing mode, the nematodes die through mass propagation, parasitize the eggs and the female nematodes of the plant parasitic nematodes, play an important role in controlling the plant parasitic nematodes under natural conditions, are one of biocontrol fungi with most potential development values (Cuo Wei Pong, Hipporen Heng, Liu xing loyalty, biological control brief introduction of root knot nematodes [ J ]. proceedings of Guizhou academy of agriculture, 1996,15(2):51-55.), and have wide adaptability, are easy to culture, can parasitize various plant nematodes, and are favorable for field control. Meanwhile, the nematicide as a nematode biocontrol bacterium substitute for chemical pesticides is already popularized and applied in some countries, and achieves some achievements. However, due to the complexity of the soil ecosystem, especially the soil bacteriostasis, the application of biocontrol bacteria is limited.

The space mutation breeding technology is an effective new mutation breeding technology and has shown important roles in creating specific mutant gene resources and breeding new crop varieties. It has been reported that China Nakan eight airship carries beauveria bassiana separated from monochamus alternatus larva, a mutagenic strain with high insecticidal speed and high pathogenicity is screened out, and the monochamus alternatus has great application potential in forest prevention and control of monochamus alternatus (Wangxing, Wang, Majiawei, Guowei, Liuhongjian, Dong Guangping. screening of beauveria bassiana high-toxicity aerospace mutagenic strain [ J ]. insect bulletin, 2014, (11): 1299. 1305.); meanwhile, after the eight spaceship of the Shenzhou carries the plant nematode biocontrol bacterium paecilomyces lilacinus Shandong strain, the biological characteristics of the mutagenic strain are differentiated to different degrees from the original strain, and excellent strains (Wangyuan, Wang, Xiwang, Zhutianhui, biological effect of spaceflight carrying the paecilomyces lilacinus [ J ]. Nuclear agriculture report 2014, (11): 1933) with large-amplitude positive variation in the aspects of growth characteristics and pathogenicity are screened out. Therefore, the mutation rate of the space mutation is high, the mutation types are rich, the beneficial mutation is increased, more opportunities are provided for breeding excellent strains, and a new way is provided for breeding the excellent strains which meet the production requirements and have strong stress resistance. As a new effective mutation breeding technology, the aerospace mutation breeding technology needs to detect various characters after aerospace mutation treatment to judge the effect of aerospace mutation and screen to obtain a target variety with specific variation (agriculture group, Zhang Yang Hua, Hupan, Gaussong, Zhang Gift. aerospace mutation has biological effect on entomopathogenic fungi [ J ] fungus article, 2006,25(4):674 and 681.).

As a biological pesticide, the chlamydosporia has many advantages which are incomparable with chemical pesticides like other plant nematode biocontrol bacteria, but also has the defects of slow growth, large environmental influence, unstable control effect, poor self-resistance and the like, so that the nematode biocontrol fungus chlamydosporia needs to be subjected to space mutation, and a mutagenic strain with high spore yield, strong nematode killing capacity and good pesticide resistance is screened out, so that the effect of controlling the plant nematodes is better exerted.

Disclosure of Invention

The invention mainly aims to carry out space mutation on Pochonia chlamydosporia (Pochonia chamydosporia) so as to screen and obtain a mutant strain with good salt tolerance and high pathogenic capability to root-knot nematode eggs;

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

The above object of the present invention is achieved by the following technical solutions:

the invention carries the cultured and activated chlamydomonas chlamydosporia into space, takes the original chlamydomonas chlamydosporia strain which is not carried as a contrast after the space mutation is finished, and compares the variability of the space mutation strain in the aspects of colony morphology, pigment change, spore morphology, growth speed, sporulation quantity, pathogenicity, salt tolerance, benomyl resistance and the like. The invention discovers that abundant character variation is generated after space mutation of Pochonia chlamydosporia. In general, space mutation shows different variation trends and amplitudes for various characters such as growth speed, sporulation quantity, pathogenicity, salt tolerance, benomyl resistance and the like. Growth speed, sporulation amount or pathogenicity and the like are important indexes for screening biological control strains, space mutation enables the characteristics to show different variation trends and amplitudes, and proves that the space mutation is non-site-specific, wide, positive and negative bidirectional, has multiple mutation sites and high mutagenesis rate, and can cause physiological variation and genetic variation of the organism variation (agricultural group, Zhang Yang Hua, Hupan, Gaussong, Zhang Gift. space mutation has biological effect on entomopathogenic fungi [ J ] fungus article, 2006,25(4): 674-681).

In order to obtain mutant strains which have good salt tolerance and high pathogenic capability to root knot nematode eggs and are suitable for biological prevention and control by screening, the invention carries out biological detection on the primarily screened chlamydosporia aerospace mutagenesis strains, such as morphology, pigment, hypha and spore morphology, colony growth speed, hypha dry weight, sporulation quantity, pathogenicity and the like, and measures the salt tolerance and the resistance to benomyl of each aerospace mutagenesis strain:

the chlamydomonas chlamydosporia aerospace mutant strain produces obvious variation on the pathogenicity of meloidogyne incognita. The parasitism rate of the mutant strain Pc-m-130 to the southern root knot nematode eggs is 94.30%, compared with the parasitism rate of the original strain to the southern root knot nematode eggs, the parasitism rate is obviously improved, and the higher parasitism rate has important significance for field biocontrol practice.

The strain after the space mutation has different types of variation on the salt tolerance. The first type is that the growth rate is accelerated along with the increase of the salt concentration, the growth rate is slowed down along with the increase of the salt concentration after reaching a certain value, and the optimum growth salt concentration is 0.10-0.15 mol/L. The second type is a slow growth rate with increasing salt concentration. The variance analysis shows that the difference between the tolerance of Pc-m-10 to a high-salt environment (0.50mol/L) and the original strain Pc is obvious (P is less than 0.05), and the mutant strain can stably grow in the high-salt environment, which indicates that the mutant strain has stronger stress resistance and can stably survive in saline-alkali soil for a long time.

The mutant strain after space mutation is subjected to benomyl resistance screening, and the screening result shows that the mutant strain has different variations on benomyl resistance, wherein the mutant Pc-m-6 has better resistance to benomyl, the difference between the resistance to benomyl (30 mu g/mL) and the original strain Pc is obvious (P is less than 0.05), and the mutant Pc-m-10 can stably grow under the condition of the benomyl concentration. In practical application, the prevention and control of root-knot nematodes are mainly based on chemical agents, and the application of the chemical agents seriously influences the growth and reproduction of soil microorganisms and even endangers beneficial microorganisms (Orthodes, Wangjing, Nicotiana tabacum root-knot nematode disease research progress [ J ]. Shenyang agriculture university school news, 2001,32(3): 232-. Therefore, the drug resistance of the biocontrol bacteria is improved while the biocontrol bacteria control effect is improved, and the biocontrol bacteria can be effectively used for disease control (wish brightness, Zhang Du, Li flying day, and the like. biological control research on the tobacco root knot nematode is advanced [ J ]. microbiological bulletin, 2004, 31 (6): 95-99.). The chlamydosporia aerospace mutant Pc-m-6 has good resistance to benomyl (the final concentration is 30 mu g/mL), which indicates that the strain has good compatibility with common bactericides such as benomyl and the like, and is very favorable for popularization and application of the chlamydosporia in production.

Finally, the mutant strain Pc-m-130 with excellent performance and suitable for biological control is obtained by screening from a plurality of space mutant strains, can stably and quickly grow in a high-salt environment, has high pathogenic capability to root-knot nematode eggs, and has important application prospect in the prevention and control of the root-knot nematodes as a biological control microbial inoculum.

The invention submits and deposits Pochonia chlamydosporia (Pochonia chamydosporia) aviation body mutation mutant strain Pc-m-10, and the microorganism preservation number is as follows: CGMCC No. 12514; and (3) classification and naming: pochoniamychamydospora chlamydosporia; preservation time: 2016, 6 months, 14 days; the preservation unit is: china general microbiological culture Collection center; the preservation address is: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

The invention further provides a biocontrol agent for controlling root-knot nematodes, which comprises: spore powder or fermentation liquid of Pc-m-130 of Pochonia chlamydosporia (Pochonia chamydosporia) mutant strain and carrier with effective preventing and treating amount.

For reference, the Pc-m-130 spore powder of Pochonia chlamydosporia (Pochoniacamydosporia) mutant strain can be prepared by the following method by the skilled person:

(1) preparing Pc-m-130 fermentation liquid of the chlamydosporia mutant strain; (2) spore products are recovered from the Pc-m-130 fermentation liquid of the chlamydosporia mutant strain; (3) drying the recovered Pc-m-130 fermentation product of the Pochonia chlamydosporia mutant strain to obtain spore powder.

The skilled in the art can prepare the chlamydomonas chlamydosporia fermentation liquor according to various methods disclosed in the literature; for example, a three-stage culture method is employed, namely: seed culture, secondary liquid amplification culture and liquid fermentation culture to prepare the Pc-m-130 fermentation liquid of the chlamydosporia spaceflight mutant strain; wherein, during liquid fermentation culture, fermentation can be carried out by adopting a fermentation tank or a shake flask culture mode; the culture medium and the culture conditions used in the three-stage culture are disclosed in the literature and are used as references, wherein the liquid fermentation medium comprises a carbon source and a nitrogen source; the char source includes, but is not limited to, any one or more of glucose, sucrose, maltose, soluble starch, or corn meal. The nitrogen source includes, but is not limited to, any one or more of sodium nitrate, ammonium sulfate, peptone or soybean meal.

The invention further provides a method for preparing the biocontrol agent of the Pochonia chlamydosporia for preventing and treating the root-knot nematode, which comprises the following steps: mixing Pc-m-130 spore powder or fermentation liquid of the spaceflight mutation mutant strain of the chlamydosporia with a carrier, uniformly stirring, crushing and sieving to prepare a corresponding microbial preparation, such as a granular preparation, a powder, a wettable powder or a microcapsule microbial inoculum and the like.

Wherein, the carrier can be diatomite, kaolin, wood chips, activated carbon, grass carbon, crop straws, dry farmyard manure and the like; in addition, auxiliary materials or/and auxiliary agents can be added into the chlamydomonas microbial preparation; the auxiliary materials can be crab shell powder or chitin and the like; the auxiliary agent can be a wetting agent, a dispersing agent or a stabilizing agent and the like.

Drawings

FIG. 1 is the front, back and side morphological pattern of colonies of Spinochonia ovirens induced by aerospace;

FIG. 2 is the spore morphology of Sporosporium chlamydomonas induced by aerospace;

FIG. 3 is a bar graph of the colony growth rate of Spaceflight-inducing Mega chlamydosporia;

FIG. 4 is a bar graph of the dry weight of hyphae of space-induced thick Pochonia chlamydosporia strains;

Detailed Description

Embodiments of the present invention will be more specifically described by the following examples, but it should be understood that the examples are merely illustrative and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Example preparation of wettable powder of Pochonia chlamydosporia

Preparation of chlamydosporia spore powder

Preparation of fermentation broth

1. Activating the chlamydosporia aerospace mutagenesis mutant strain, and then performing shake flask seed culture;

composition of shake flask seed culture medium (in mass percent):

1.5 to 2 percent of glucose

0.4 to 0.6 percent of sodium nitrate

0.2 to 0.3 percent of ammonium sulfate

0.2 to 0.3 percent of soybean meal

Dipotassium hydrogen phosphate 0.1-0.2%

Magnesium sulfate 0.1-0.2%

Balance of water

Liquid fermentation conditions: 200ml of seed culture medium is filled into a 500ml triangular flask, the chlamydosporia spore suspension is inoculated after autoclaving, the inoculum size is 1.5-3%, the mixture is placed at 24-28 ℃, the shaking table is used for 150-.

2. Secondary liquid scale-up culture (by mass percent):

the culture medium for the secondary liquid amplification culture comprises the following components:

3 to 5 percent of glucose

0.4 to 0.6 percent of sodium nitrate

0.2 to 0.3 percent of ammonium sulfate

0.2 to 0.3 percent of soybean meal

Dipotassium hydrogen phosphate 0.1-0.2%

Magnesium sulfate 0.1-0.2%

Balance of water

Sterilizing the secondary liquid amplification culture medium in situ in a fermentation tank;

liquid fermentation conditions: the fermentation temperature is 25-28 ℃, the tank pressure is 0.6-0.8bar, the oxygen introduction amount can be 300L/h at 100-.

3. Liquid fermentation culture:

the culture medium for liquid fermentation culture comprises the following components:

10 to 18 percent of glucose

0.2 to 0.3 percent of sodium nitrate

0.1 to 0.15 percent of ammonium sulfate

0.1-0.15% of soybean flour

Dipotassium hydrogen phosphate 0.1-0.2%

Magnesium sulfate 0.1-0.2%

10-30% of potato extract (20%)

Balance of water

Sterilizing the liquid culture medium in situ in a fermentation tank;

liquid fermentation conditions: the fermentation temperature is 28-30 ℃, the oxygen introduction amount can be 300L/h of 100-; initial pH: 5.0-6.0; rotating speed: 150-; tank pressure: 0.6-0.8Mpa, fermentation period controlled at 6d, and spore concentration up to 10 under microscopic examination⁹The dosage is more than one per ml.

(II) recovering and drying the spore product from the fermentation broth

Centrifuging the fermentation liquor under the following centrifugation conditions: the relative centrifugal force is 4000g, the centrifugal time is 40min, and 2.0g of flocculating agent is added into every 100 ml of fermentation liquor; and (4) discarding the supernatant, adsorbing the precipitate bacterium slurry by using a diatomite carrier, and drying in the shade until the water content in the product is lower than 10%, thus obtaining the chlamydosporia spore powder.

Preparation of wettable powder

Weighing the following components in percentage by mass: 85% of the prepared spore powder, 11% of diatomite, 2% of wetting agent PEG and 2% of dispersant sodium lignosulphonate; uniformly mixing spore powder and diatomite, crushing and sieving with a 325-mesh sieve; and uniformly mixing the crushed product with a wetting agent and a dispersing agent, grinding the mixture by using an airflow mill, and uniformly mixing the mixture to obtain the composite material.

Test example 1 screening of chlamydosporia navicula mutant strain Pc-m-6 and test for measuring mutagenesis Effect

1 materials and methods

1.1 biological Material

The test strain Pochonia chlamydosporia (P.chamydosporia) originates from the United states and is a production strain for preventing and treating root-knot nematodes, and the strain is stored in the forest ecological environment and protection institute of China forestry science research institute. After the space mutation is returned to the field, the sample is stored at 4 ℃ in the laboratory of the inventor and is ready for use. The southern root knot nematode (m.incognita) to be tested was provided by the institute for forest ecology and protection, national institute for forestry and science.

1.2 space flight carrying

The mycelia mass activated for 7 days by PDA culture were transferred to 2 sterile EP tubes (1.5mL) and sealed. One tube was spaceflight mounted (shenzhou eight), and the other tube was stored in a 4 ℃ refrigerator as a control material.

1.3 mutant screening

Respectively preparing chlamydosporia subjected to space mutation treatment and an original strain into spore suspensions, and diluting the spore concentration to 1.0 x 10³cfu/mL, evenly coating the mixture on a PDA (personal digital assistant) plate with the diameter of 9cm (0.1 mL of spore suspension is added in each dish), culturing the mixture at the constant temperature of 25 ℃ for 6d to 9d, observing the size, the shape and the front and back colors of a single colony, and selecting a single colony strain which is obviously different from the original strain; the original number is "Pc", and the remaining strains are numbered "Pc-m-No.".

1.4 detection of the effects of space mutagenesis

The variability of the aerospace mutant strain in colony morphology, pigment change, spore morphology, growth speed, sporulation amount, pathogenicity, salt tolerance, benomyl resistance and the like is compared by taking an original non-carried chlamydia chlamydosporia strain as a control.

1.4.1 morphological and pigment Change observations of colonies

3 colonies were cut from a well-grown PDA plate by a punch having a diameter of 5mm in 5mL of a solution containing 0.5% Tween, and sufficiently shaken to prepare a spore suspension. 2.5. mu.L of spore suspension (spore concentration 1X 10) was inoculated into the center of a 9cm dish (15mL of medium)⁶cfu/mL), incubated in an incubator at 25 ℃ and each strain was repeated 3 times.

1.4.2 Observation of spore morphology

Suspending the spores (spore concentration 1X 10)⁶cfu/mL) were dropped on a glass slide to make a provisional slide, and spore morphology was observed under a microscope.

1.4.3 colony growth Rate determination

A sterilized 5 mm-diameter filter paper is dipped in a spore suspension (spore concentration 1X 10)⁶cfu/mL) was placed in the center of a PDA medium plate, cultured in an incubator at 25 ℃ and the colony diameter was measured every 3d by the cross method until the strain grew over the entire dish. The original strain was a control and was replicated 3 times.

1.4.4 hypha Dry weight determination

150mL conical flask containing 50mL PD medium, 200. mu.L of suction (spore concentration of 1X 10)⁶cfu/mL) spore suspension in each flask of culture medium, and the temperature of the constant temperature shaking table is 25 ℃ and the rotating speed is 120 r/min. After 96h of culture, pouring all bacteria liquid in the triangular flask into a 50mL centrifuge tube, centrifuging for 3min at 5000r/min, pouring out supernatant, pouring sediment into the centrifuge tubeDrying on filter paper in an oven at 50 ℃ to constant weight, and weighing.

1.4.5 pathogenicity assay

Collecting diseased roots in a greenhouse, cleaning the diseased roots, cutting the roots into small sections of 0.5cm, filling the small sections into a 500mL triangular flask, pouring 200mL of 1% sodium hypochlorite solution, sealing the triangular flask, shaking for 3min, quickly sieving with a 200-mesh sieve, quickly sieving with the 500-mesh sieve, repeatedly washing eggs left on the 500-mesh sieve with distilled water, finally washing with sterile water, collecting in a sterile small beaker, observing under a microscope, and adjusting the concentration of the eggs to 1000/mL. mu.L of egg suspension was added to a 6cm diameter petri dish, and 2mL of 1.0X 10⁶one/mL mutant spore suspension, wild type Pc as control. Each mutant strain is repeated for 3 times, cultured for 5 days at 25 ℃, the number of parasitic eggs and the number of non-parasitic eggs are counted, and the parasitic rate of the strain spores to the meloidogyne incognita eggs is calculated.

1.4.6 determination of salt tolerance

PDA culture medium containing NaCl 0.05mol/L, 0.10mol/L, 0.15mol/L, 0.20mol/L, 0.25mol/L and 0.50mol/L is prepared. Dipping the spore suspension of the space mutation mutant strain (the spore concentration is 1 multiplied by 10) by a sterilized filter paper sheet with the diameter of 5mm⁶cfu/mL) was placed in the center of the above PDA medium containing NaCl at various concentrations, cultured in an incubator at 25 ℃ and the colony diameter was measured after 9d and the growth rate was calculated.

1.4.7 benomyl resistance Screen

Preparing a PDA culture medium with a final concentration of 30 mug/mL benomyl (benomyl specification is 50% wettable powder, a product of Jiangsu Lanfeng biochemical industry Co., Ltd.). Dipping the spore suspension of the space mutation mutant strain (the spore concentration is 1 multiplied by 10) by a sterilized filter paper sheet with the diameter of 5mm⁶cfu/mL) was placed in the center of PDA medium at a final concentration of 30. mu.g/mL benomyl, cultured in an incubator at 25 ℃ and the colony diameter was measured after 9 days and the growth rate was calculated.

1.4.8 data processing and analysis

The data were processed using Microsoft Excel software. One-way ANOPC-M-a was performed using SPSS 19.0 and the different strains were compared multiply using the Duncan method (P0.05) and the experimental daA are presented as mean plus minus standard deviation (mean ± SD, n 3).

2 results and analysis

2.1 Effect of space mutagenesis on colony morphology and pigment changes

After the space-induced chlamydomonas is subjected to 3-4 generations of subculture to obtain stable characters, the colony is observed to have obvious morphological differentiation (figure 1), and differentiated strains can be divided into 4 types of types I, II, III and IV according to the morphological and color changes of the front, back and side of the colony: the morphology of the I-type bacterial colony is similar to that of the original bacterial strain Pc, the front of the bacterial colony is white down, the back of the bacterial colony is light yellow, the side surface of the bacterial colony is smooth and arched, and the total 11I-type bacterial colonies account for 37.9 percent of the aerospace mutagenic bacterial strain; the front side of a colony of the II type bacterial strain is white down, the center of the colony is sunken and has folds, the back side of the colony is light yellow and is darker than the I type, and the number of the II type bacterial colonies is 14, and the II type bacterial colonies accounts for 48.3% of the aerospace mutagenesis bacterial strain; the front surface of the III type bacterial colony is white down, the center of the bacterial colony is sunken without folds, the back surface of the III type bacterial colony is light yellow, 3 III type bacterial colonies are respectively Pc-m-13, Pc-m-26 and Pc-m-54, and account for 10.3 percent of the space mutation bacterial strain; the front surface of the IV-type bacterial colony is white down, the center of the bacterial colony is white and convex and has folds, the back of the bacterial colony is yellow, and the IV-type bacterial colony only has one strain Pc-m-9 and accounts for 3.4 percent of the space mutation strain.

2.2 Effect of space mutagenesis on spore morphology

Observed by a microscope (100 mu m), the conidia of the space-induced thick chlamydosporia is spherical or nearly spherical, transparent and smooth, and the four types (I, II, III and IV) have no obvious difference with the original strain Pc.

2.3 Effect of space mutagenesis on colony growth Rate

According to observation, the growth speed of the space-induced thick chlamydomonas is about 1cm/3d, but the growth speed becomes slow after 15d, and the bacterial colony can grow over the whole dish after about 20 d. The bacterial colony diameter of the cultured 15d strain is more than or equal to 10 strains including Pc-m-10 of the original strain, and accounts for 34.5 percent of the mutant strain; the strains smaller than the original strain Pc were 19 strains, accounting for 65.5% of the mutant strains. Analysis of variance showed that the mutant strain Pc-m-38 grew faster at the first 9d, differed significantly from the original strain Pc (P <0.05), and then grew at a progressively slower rate (Table 1, FIG. 3).

TABLE 1 colony growth rate of space-induced thick chlamydomonas

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

Note: data in the table are mean ± sd, followed by a significant difference compared to the original strain Pc (P <0.05). Note, Data in the table is mean + -SD, Data marked-by-representations signature (P <0.05).

2.4 Effect of space mutagenesis on hyphal Dry weight

The hypha dry weight of the space mutation strain has positive and negative bidirectional variation in the culture process (figure 4). Compared with the original strain Pc, 13 strains with improved dry weight of hyphae account for 44.8 percent of the mutant strain; the dry weight of hyphae was reduced by 16 strains, accounting for 55.2% of the mutant strains. Wherein the dry weight of Pc-m-26 mycelium is 0.2776 g; the mutant strain with the smallest dry weight was Pc-m-27, which was 0.1559g in dry weight. The dry weight of the hyphae of Pc-m-130 is obviously improved compared with the dry weight of the original strain, and the variance analysis shows that the hyphae is obviously different from the original strain (P <0.05).

2.5 Effect of space mutagenesis on pathogenicity

The test results are shown in Table 2. The chlamydomonas chlamydosporia aerospace mutant strain produces obvious variation on the pathogenicity of meloidogyne incognita. Compared with the original strain Pc, 20 strains with improved parasitism rate to Meloidogyne incognita eggs account for 69.0% of the mutant strain; the number of strains with reduced parasitism was 9, accounting for 31.0% of the mutant strains. Wherein, the parasitism rate of the mutant strain Pc-m-130 to the meloidogyne incognita eggs is 94.30%, which is obviously improved compared with the original strain, and the difference between the mutant strain and the original strain is obvious (P is less than 0.05).

TABLE 2 parasitism rate of Sporoselaginella chlamydosporia to Meloidogyne incognita

Table 2 Parasitic rate of aerospace mutant strains of Pochoniachamydosporia against Meloidogyne incognitaeggs/％

2.7 Effect of space mutagenesis on salt tolerance

The test results are shown in Table 3. The strain after the space mutation has different types of variation on the salt tolerance. The first type is that the growth rate is accelerated along with the increase of the salt concentration, the growth rate is slowed down along with the increase of the salt concentration after reaching a certain value, the optimum growth salt concentration is 0.10-0.15mol/L, the number of the mutant strains is totally 17, the mutant strains account for 58.6 percent of the mutant strains, and Pc-m-6 and the original strain Pc also belong to the first type. The second type is a slow growth rate with increasing salt concentration; the number of the mutant strains is 9, and the mutant strains account for 31.0 percent of the mutant strains. In addition, 3 mutant strains are not in the two types, namely Pc-m-30, Pc-m-39 and Pc-m-51, and account for 10.3 percent of the mutant strains. Analysis of variance shows that the tolerance of Pc-m-130 to high-salt environment (0.50mol/L) is different from that of original strain Pc (P <0.05), and the mutant strain can keep higher growth rate and grow stably in the high-salt environment.

TABLE 3 salt tolerance assay for space-induced thick chlamydomonas

Table 3 Salt tolerance of aero space mutant strains of Pochoniachamydosporia/cm

2.8 Effect of space mutagenesis on benomyl resistance

The results are shown in Table 4, and after space mutation, the strain shows different variations of benomyl resistance (Table 4). The final concentration of the benomyl in the culture medium is adjusted to be 30 mug/mL, and the resistance of the mutant Pc-m-130 to the benomyl (30 mug/mL) is not obviously different from that of the original strain Pc by the observation result after the benomyl is cultured for 9 days at 25 ℃.

TABLE 4 benomyl resistance screening of space-induced thick chlamydosporia

Table 4Benomyl resistance of aerospace mutant strains of Pochoniachamydosporia/cm

Experimental example 1 field experiment of wettable powder prepared from Pochonia chlamydosporia space mutation mutant strain Pc-m-130 for preventing and treating root-knot nematode of Zanthoxylum bungeanum

Test materials and methods

1.1 test inoculum

Test bacteria agent: wettable powder prepared by using the chlamydosporia spaceflight mutagenesis mutant strain Pc-m-130 (prepared in example 1);

comparison with microbial inoculum: wettable powders (prepared as in example 1) were prepared with the original strain of Pochonia chlamydosporia (not subjected to space mutagenesis).

1.2 Experimental crops

And (4) planting pepper trees in the pepper planting area.

1.3 design of the experiment

The experimental site is arranged in a pepper planting area with large and uniform nematode worm quantity, and 20-30 heads of the second-instar larvae of the root-knot nematodes are investigated in each 100g of soil before medicine application.

The experiment is divided into 3 groups, experiment 1 group, experiment 2 group and clear water control group, and the specific experiment design is as follows:

test 1 group was treated with the wettable powder prepared in example 1;

control group 2 was treated with control inoculum.

Clear water control group: adopting clear water for treatment (no any medicament for preventing and treating the nematodes is applied) and not performing nematode prevention treatment;

each treatment was 30 replicates (in three columns of 10 pepper trees), each treatment was randomized.

And (3) performing a microbial inoculum plot test from 4 months 15 days to 5 months 10 days, digging a soil layer with the thickness of 10cm and the radius of 50cm on the surface of the root around the pepper tree, diluting 1ml of original microbial inoculum (the test microbial inoculum and the reference microbial inoculum are the same in amount and are 1ml of original microbial inoculum/plant) with water, uniformly spraying the diluted microbial inoculum in the dug pit in a spraying mode, distributing the microbial inoculum around the root, and resetting the dug soil. The root knot index (disease index) of the pepper tree and the number of second-instar larvae in soil are investigated after 90 days of pesticide application, and the control effect is evaluated.

The root node index (disease index) calculation method comprises the following steps: the roots of each treated prickly ash are dug out and investigated according to the grading survey index of root-knot nematodes, the root-knot severity is graded from 0 to 10, and the grading standard refers to Benjamin et al (Benjamin D, Grover C BJ. the grading of compatible and compatible responses of position to Meloidogyneic origin. journal of neurology, 1987, 19: 218-). The root node index calculation formula is as follows:

percent reduction of larvae (relative fall rate) calculation method: soil samples of 5 points per plot were collected from the crop root circumference (0-20cm depth) using a soil drill (2 cm. times.H 20cm), mixed well, 100g of the soil samples were separated by centrifugal floatation method (Karsen G. the plant parasitical Nematode Gen Meloidogyne Godi, (Tylenchida) in Europe [ M ] Gent: Drukkeru Modern,1982:5-24.), fixed with 4% formalin after heat-killing, counted under an inverted microscope, and the number of root knot Nematode second instar and the percentage of reduction of larvae (relative reduction) in 100g of the soil samples were calculated. The calculation formula is as follows:

the relative prevention and treatment effect calculation formula is as follows:

2 results of the experiment

The results are shown in Table 5.

TABLE 5 experiment on the prevention and treatment effect of Pc-m-130 wettable powder of chlamydosporia aerospace mutagenesis mutant strain on Meloidogyne Zanthoxylum

As can be seen from the test data in Table 5, the relative control effect of the test bacterial agent (wettable powder prepared by the spaceflight mutagenesis mutant strain Pc-m-130 of the Pochonia chlamydosporia) on root-knot nematodes is 18.75 percent higher than that of the control bacterial agent (wettable powder prepared by the original strain of the Pochonia chlamydosporia), and the larva reduction percentage of the test bacterial agent is 17.3 percent higher than that of the control bacterial agent. The test result of field root-knot nematode prevention and control shows that the biological prevention and control effect of the chlamydosporia spaceflight mutagenesis mutant strain Pc-m-130 on the root-knot nematode is far better than that of the original strain on the root-knot nematode.

Claims

1. A Pochonia chlamydosporia (Pochonia chamydosporia) space mutation mutant strain Pc-m-130 is characterized in that the microorganism preservation number is as follows: CGMCC No. 12514.

2. Use of the chlamydosporia overtraving mutagenesis mutant strain Pc-m-130 as claimed in claim 1 for the control of root-knot nematodes.

3. A biocontrol agent for controlling root-knot nematodes comprising: spore powder or fermentation liquid of the space mutation mutant strain Pc-m-130 of Pochonia chlamydosporia as claimed in claim 1, and a carrier.

4. The biocontrol agent according to claim 3, characterized in that: the carrier is diatomite, kaolin, wood chips, active carbon, grass carbon, crop straws or dry farmyard manure.

5. The biocontrol agent according to claim 3, characterized in that: also contains auxiliary materials or auxiliary agents.

6. The biocontrol agent according to claim 5, characterized in that: the auxiliary material is crab shell powder or chitin; the auxiliary agent is a wetting agent, a dispersing agent or a stabilizing agent.

7. A method of preparing the biocontrol formulation of claim 3 comprising the steps of: (1) culture of the spaceflight mutagenesis mutant strain Pc-m-130 of Pochonia chlamydosporia according to claim 1 to obtain spore powder or fermentation liquid; (2) mixing the obtained spore powder or fermentation liquid and the carrier, stirring uniformly, pulverizing and sieving to obtain the biocontrol agent.