CN114317293B - High pathogenicity and high ultraviolet resistance space insect fungus strain SCAUHT18 for common thrips and application thereof - Google Patents
High pathogenicity and high ultraviolet resistance space insect fungus strain SCAUHT18 for common thrips and application thereof Download PDFInfo
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Abstract
The invention relates to the field of biological control, in particular to an excellent space bacterial strain with high pathogenicity and high ultraviolet resistance to common thrips, which is beauveria bassiana @Beauveria bassiana) The strain SCAUHT18 is deposited with the Guangdong province microorganism strain collection center (GDMCC) with the deposit number of GDMCC NO. 62188. The invention combines the toxicity and ultraviolet resistance of the original strain and the space mutation strain to common thrips, the indexes of strain screening, reasonable experimental scheme and the like into comprehensive consideration. The strain with excellent pathogenicity and high ultraviolet resistance to common thrips is screened, and the strain has great potential for biological control of common thrips.
Description
Technical Field
The invention relates to the field of plant protection, in particular to the field of biological control, and especially relates to the biological control of common thrips.
Background
With the development of sustainability, the protection of the ecological environment is particularly important. In the development process of agricultural technology, the unreasonable application of pesticides easily causes the formation of various ecological environment pollution problems. Chemical control is usually adopted in production, so that not only is the effect not ideal, but also a series of problems such as drug resistance, massive death of natural enemy insects, loss of biodiversity, exceeding of pesticide residues, ecological environment pollution and the like are caused by pests (Yang Anjun. Harm of pesticides and discussion of green plant protection technology [ J ]. Agricultural development and equipment, 2021 (04): 132-133). With the sustainable development of agricultural economy in recent years, the use amount of pesticides is continuously increased, the amount of pesticides used for preventing and controlling crop diseases and insect pests is about 600 ten thousand t each year, the effective utilization rate of the pesticides is only 30%, and the remaining 70% of the pesticides belong to the loss amount (He Yunfeng. Harm of pesticides and research on green plant protection technology [ J ]. Agricultural technologies and equipment, 2021 (01): 94-95). The problem of pesticide pollution is more serious and even pesticide poisoning cases occur frequently due to the continuous expansion of the pesticide usage (Liu An. Pesticide environmental hazard problem and countermeasure research [ J ]. Green science and technology 2020 (12): 158-160.).
At this time, a conscious protection technical mode for strengthening green plants is needed, so that the prevention and control work of pesticide pollution problem is reduced as much as possible. Therefore, the traditional prevention and control mode using chemical pesticides mainly is changed, the plant protection policy of 'pre-prevention mainly and comprehensive prevention and control' is implemented, the sustainable development of sustainable health for comprehensively treating pests by adhering to the basis of agricultural prevention and control mainly and biological prevention and control and combining other prevention and control measures has extremely important significance (Yang Yunhua, du Kaishu, dan Mingwang. Biological prevention and control research progress of entomogenous fungi [ J ]. Henan academy of technology, 2011, 39 (1): 34-37).
Entomogenous fungi are a type of parasitic fungi that enter the insect body through infestation and absorb insect nutrition, ultimately leading to death of the host. The chemical pesticide has different action modes, is safe to the environment and human body, and is commonly used for biological control of pests. There are two main ways in which entomogenous fungi infest insects: external routes, which are in contact with insects, invade from body walls, valves, wounds, etc.; the internal route is invaded through the digestive tract and respiratory tract when the insect feeds on and breathes. External invasion is exemplified by surface invasion, firstly, the parasitic fungus adheres to the body surface by conidium, then the conidium germinates to grow into a bud tube, the bud tube penetrates through the body wall of the host and enters the blood cavity of the host, and the inside of the host is rapidly propagated in the form of mycelium or through small independent propagules, so that the insect nutrition is absorbed in haemolymph and toxic substances are continuously generated and released, and finally the death of the host insect is caused (Li Yue, jiang Chunjie, zhao Yuying, and the like; the application of the parasitic fungus in biological control of forestry pests [ J]Plant protection 2016, 10 (20): 10-24). The internal invasion is achieved byEntomogenous fungi invade through the insect digestive tract, respiratory tract and the like, then germinate and grow in the host body until the nutrition in the insect body is consumed. At present, the reported entomogenous fungi in China have more than 400 species, namely, destruxins of Metarrhizium anisopliaeMetarhiziumspp and Beauveria bassianaBeauveriaspp is the most widely commercially available. Beauveria bassiana (Beauveria) BeauBeauveriaThe host range is wide, including important agricultural and forest pests, disease vector insects and acarids, is easy to culture, is harmless to warm-blooded animals and plants, has strong disease curing ability to adults, can infect larvae, pupae, adults and other insect states, has continuous effect on the next generation and plays an important role in the management of agricultural and forest pests. The entomogenous fungi are well known for the advantages of multiple species, safety, effectiveness, long application period, no harm to natural enemies, difficult resistance generation, rapid mass production and the like, and have incomparable repeated infectivity and production convenience in biological control (Wang Qinghai, wanping, yellow jade Jie, and the like; the application research of the entomogenous fungi in biological control of pests [ J)]Shandong science 2005, 18 (4): 37-40.).
The space breeding is a high and new technology breeding way and method for breeding new plant germplasm and new material and new variety on the ground by utilizing the space environment which can be reached by return satellite and the like to generate beneficial mutation on the mutagenesis action of plants (seeds), which is called space mutation breeding or space mutation breeding (Ma Cheng, ma Weichao, an Jianping, li Shiweng. Application of Chinese microorganism space mutation breeding and research progress [ J)]Hunan university of agriculture 2012 (19): 5-8.). The unique conditions of the space environment, including ultra-high vacuum, ultra-clean, microgravity, strong radiation, and a large difference from the ground conditions, and also strong ultraviolet irradiation, etc., may cause genetic variation of organisms. Space environment is a special important field of space science research. High mutation frequency, broad mutation spectrum, large mutation amplitude, and stable mutation character after mutation, thereby shortening breeding period and improving biological safety (Lionheart G, vandenblank J P, hoeksema J D, et al The impact of simulated microgravity on the growth of different genotypes of the m)odel legume plant Medicago truncatula[J].Microgravity Science and Technology, 2018, 30 (4): 491-502.). The space mutation of the microorganism provides a new way for the mutation breeding of the microorganism, and the space mutation can obviously change the growth characteristics of the microorganism, and is expressed in the aspects of growth speed, growth morphology, growth characteristics and the like. The space mutation provides a new way for genetic improvement of the biological control excellent strain, and the entomogenous fungi can possibly improve the strain toxicity and strengthen the stress resistance of the strain to the environment under the action of space environment through space carrying, so that the efficient engineering bacteria are obtained. These altered traits alter the ability of the microorganism to produce metabolites. Compared with the traditional mutation breeding under the same condition, the space mutation breeding has obvious effect, better genetic stability of the character and great application potential, and can bring great economic benefits (Jiang Peixia, zhang Ruiping, wang Haisheng, zodiac trace, poplar, the space mutation effect of the engineering bacteria of the violacein synthesis and the screening of high-yield strains thereof [ J]Chemical journal 2010,61 (02): 455-461.).
Common thripsMegalurothripsusitatus(Bagrall), also known as Soy thrips, soy flower thrips, is of the order ThysanopteraThysanopteraThrips subfamilyThripina（Stephens）PriesnerThrips genusMegalurothripsInsects which are widely distributed in the tropical regions of the world (Han Yunfa. Fifty-fifth book of economic insects in China: thysanoptera [ M ]]Beijing: science publishers, 1997:39-59.). At present, the prevention and treatment of thrips also mainly depend on chemical prevention and treatment means. The insect omnivorous insect pest is not fully counted, and the host plants have 9 kinds of 28 kinds, 16 kinds of which are leguminous plants (Miyazaki M, kudo I, iqbal A. Notes on the thrips (Thysanoptera) occurring on the soybean in Java [ J)].library wur, 1984, 52 (4): 482-486.). The plant growth is slow or stopped in severe cases, and the flowers and fruits are favored to be eaten. However, because thrips are small, hidden and strong in fertilityThe development of thrips has led to the continuous enhancement of drug resistance of thrips in recent years, and the chemical control means are difficult to achieve good control effect (Tang Guowen, xinwen, meng Guoling. Research on vegetable thrips species in Wuhan region [ J)]University of Huazhong agriculture journal, 2002, 21 (1): 5-9.). Because of the long-term use of chemical pesticides, some pests develop strong resistance to drugs, and natural enemies of many pests are miskilled in the control process. The chemical control is relied on to cause the problems of ecological balance damage, environmental pollution and food safety of farmlands, and common thrips have different degrees of drug resistance to various common pesticides in recent years.
The entomogenous fungi are subjected to the influence of various environmental factors, such as ultraviolet irradiation, temperature, humidity and stress of other biological or non-biological factors, the interaction of the factors can influence the activity and the infectivity of spores, the large-scale application of fungal pesticides in production is restricted, and the screening of the strains with high toxicity and high stress resistance is extremely important for field production popularization.
Disclosure of Invention
The invention screens out the strain with higher ultraviolet resistance from the beauveria bassiana strain with high virulence by mutagenesis in the out-of-flight, and applies the strain to the biological control technical means of common thrips. Therefore, the invention aims to develop the ultraviolet resistance of the space mutation high virulence strain compared with the original strain, and provides the space return beauveria bassiana strain with high virulence and ultraviolet resistance to common thrips. The strain has a relatively good insecticidal effect on common thrips, and has the advantage of high ultraviolet resistance. In order to obtain an excellent strain with the biological control effect on common thrips, the inventor adopts the research on biological characteristics of the beauveria bassiana strain with high toxicity on common thrips for the original strain and the early aerospace return, and determines the survival rate of conidia under different ultraviolet time irradiation, thereby obtaining the excellent strain with strong pathogenicity on common thrips female insects and high ultraviolet resistance. The spore yield, colony growth rate, spore activity and the like of the strain under ultraviolet irradiation at different times are important indexes for screening excellent strains.
Experiments show that the ultraviolet resistance of 20 strains of the space bacterial strain with high toxicity to common thrips and the original bacterial strain can be different from each other, so that the space bacterial strain has different degrees of ultraviolet resistance. According to the experimental result of the invention, the indexes of the screening of the excellent strains are comprehensively considered, wherein the ultraviolet resistance is an important index for measuring the outdoor biological control potential of the high-virulence fungus strains, and the colony diameters, the spore yields and the spore germination rates of the spaceflight strains SCAUHT18, SCAUHT38 and SCAUHT56 are higher than those of other spaceflight strains and original strains under the irradiation of ultraviolet rays of 0h,1h, 2h, 4h and 8h, so that the ultraviolet resistance of the spaceflight strains SCAUHT18, SCAUHT38 and SCAUHT56 is more stable than those of the original strains and other tested strains.
Furthermore, the invention also provides application of the aerospace strain SCAUHT18 in outdoor biological control of common thrips, wherein the strain is preserved in the Guangdong province microorganism strain collection (GDMCC, address Guangdong province 100 th university of Mitsui, first, china, no. 59 th building 5 th building Guangdong province academy of microorganisms, and the preservation number is GDMCC NO 62188 in 2022, 1 month and 4 days. While the aerospace strains SCAUHT38 and SCAUHT56 will be filed by further patent applications.
In particular embodiments, the strain may be cultured to produce a spore fluid, or a spore-containing microbial inoculum. Preferably, the spore concentration of the strain at the time of administration is 10 6 spores/mL to 10 9 spores/mL, most preferably 10 7 spores/mL to 10 8 spores/mL.
The inventor discovers that the original strain beauveria bassiana strain HNSB110 with a certain effect on common thrips before in screening the original strain and the space strain, has particularly outstanding effect on common thrips after space mutation returns, and obviously improves biological characteristics. Biological experiments show that the space mutation strain has 90 percent (10 percent of control effect on common thrips 7 spore/mL experiments), whereas the effect of the original strain is only 60% under the same conditions; at 1X 10 7 Under the condition of spore/ml concentration liquid, SCAUHT18, SCAUHT38 and SCAUHT56 are irradiated by ultraviolet rays at different treatment time, and the colony diameter, spore yield and spore germination rate of the sporeAll were significantly higher than the other strains, and the colony diameter of 10d reached at 8h uv irradiation: 40mm, 40mm and 37.5mm, and the spore yield reaches: 2.6X10 7 /mL、2.35×10 7 /mL、2.4×10 7 /mL; the germination rate is as high as 72 h: 62.5%, 60.5%. Far higher than other strains (see experimental results and tables for specific examples). Therefore, the beauveria bassiana strains SCAUHT18, SCAUHT38 and SCAUHT56 can effectively inhibit the outdoor common thrips population, are safe to people, livestock, plants and environment, can partially replace thrips pesticide prevention and control, and have great application potential in outdoor biological control.
Drawings
FIG. 1 is a schematic diagram of the aeronautical breeding steps of the present invention.
FIG. 24 PE tubes of the mutagenized strains obtained by space breeding.
FIG. 3 colony diameter comparison of strain SCAUHT18, SCAUHT38, SCAUHT56 and wild type strain. Wherein A is a front view of the flat plate, and B is a back view of the flat plate.
Detailed Description
The following description of the invention is made by way of example and not as a limitation of the invention.
Embodiment one: space breeding
1. Test strain
HNSB110: beauveria bassiana (Beauveria bassiana) A. CinereaBeauveria bassiana) The strain is separated from forest soil in Kunming city of Yunnan province in 9 months of 2007 and is preserved in biological control center of biological control education department of agricultural university of south China.
As early experiments show that the strain grows faster and has a certain control effect on common thrips, the strain is selected for space breeding.
2. Experimental method
The original strain HNSB110 is inoculated into 4 PE pipes, the PE pipes are sealed by preservative films, and samples are collected and then packaged in an experiment box. The on-orbit test is carried out in space by launching the airship, the returning cabin is retracted, the returning cabin is opened, the test box is opened, and finally, the sample distribution is carried out (the specific operation steps are shown in figure 1).
3. Experimental conditions
(1) The carrier comprises: carrier rocket with long sign No. five B
(2) Time: 2020.5.5-2020.5.8 (on-orbit for 67 hours)
(3) Emission field: hainan Wenchang space base
(4) On-track height: 300-8000 km
(5) The environment is that: multiple crossing Van Allen radiation band (high energy particle radiation band)
4. Experimental results
The total obtained mutagenesis strains were collected into 4 PE tubes (FIG. 2), and the strains were carried back and stored in the biological control center of the university of agricultural university of south China, and were stored at 4℃for subsequent experiments.
Embodiment two: bioassay of strains
1. Test insects: common thrips were collected from Zhu Cun cowpea fields in Guangzhou, and were cultivated in the laboratory for multiple generations at a temperature of 26℃and a relative humidity of 65% with a photoperiod of L: D=12:12.
2. Test strain:
(1) HNSB110: beauveria bassiana (Beauveria bassiana) A. CinereaBeauveria bassiana) The strain is preserved in the biological control center of the university of agricultural university of south China department of biological control education and is an original wild strain.
(2) 100 single colony spores are picked by adopting the aeronautical mutagenesis strain obtained in the first embodiment, and 100 aeronautical strains are obtained. And carrying out a primary toxicity experiment of common thrips on 100 space strains, and then carrying out a toxicity biological test of common thrips on the first 20 strains with higher toxicity.
3. Experimental method
(1) Preparation of spore suspension
Culturing at 26+ -1deg.C on PDA plate for 7d, eluting spore with 0.05% Tween-80 sterile water, stirring with magnetic stirrer, shaking culturing at 180 rpm and 25deg.C for 25 min, filtering with double-layer mirror cleaning paper, counting with blood cell counting plate, measuring mother liquor concentration, and making into 1×10 7 spore/mL spore suspensionAnd (3) liquid.
(2) Toxicity determination of entomogenous fungi on common thrips
Placing the prepared spore suspension and fermentation liquor into a flat finger-type tube (15 mm multiplied by 75 mm), soaking for 2 hours, pouring out the spore suspension, and naturally air-drying the finger-type tube for later use; cutting cowpea into segments (1 cm, two ends are pore-free), immersing in spore suspension, taking out for 30s, naturally airing, putting the cowpea segments into treated finger-shaped tubes correspondingly, simultaneously inoculating 50 ends of female adult common thrips, sealing cotton, putting the cowpea segments into a climatic chamber, taking 0.05% Tween-80 sterile water as a blank control, and repeating each treatment for 4 times. Mortality was recorded for 7 days.
(3) Data processing
The SPSS 19.0 software is used for experimental treatment analysis, and the single-factor analysis of variance is used for analyzing each result and Tukey is used for checking the difference significance.
4. Experimental results
Experimental results show that the pathogenicity of spore suspensions of different strains on common thrips is obviously different, the pathogenicity of different strains at the same concentration and different treatment times is different, and the death rate of female adults of common thrips is increased along with the increase of the concentration and the treatment time (table 1); when the concentration is 1X 10 7 The cumulative mortality at spore/mL (Table 1), SCAUHT18, SCAUHT38, SCAUHT56 at 6d was 100%,100%,100%.
The results show that the strains SCAUHT18, SCAUHT38 and SCAUHT56 have better lethal effect on common thrips, and belong to potential excellent biocontrol bacteria.
TABLE 1 correction mortality of common thrips with different strains (%)
Note that different lower case letters in the same column indicate significant differences in virulence between different strains (P < 0.05) by Tukey test.
Embodiment III: anti-ultraviolet biological characteristics of aerospace high-virulence strain
1. Test strain
HNSB110: beauveria bassiana (Beauveria bassiana) A. CinereaBeauveria bassiana) Is preserved in the biological control center of the university of agricultural university of south China.
20 strains selected from the second strains. Wherein SCAUHT18, SCAUHT38, SCAUHT56 was deposited with the Guangdong province microorganism strain collection center (GDMCC) under accession numbers GDMCC NO. 62188, GDMCC NO. 62187 and GDMCC NO. 62199, respectively.
2. Test method
(1) Preparation of spore suspension
Culturing at 26+ -1deg.C on PDA plate for 7d, eluting spore with 0.05% Tween-80 sterile water, stirring with magnetic stirrer, shaking culturing at 180 rpm and 25deg.C for 25 min, filtering with double-layer mirror cleaning paper, counting with blood cell counting plate, measuring mother liquor concentration, and making into 1×10 7 spore/mL spore suspension.
(2) Ultraviolet irradiation
Will be 1X 10 7 The spores/mL spore suspension was irradiated with ultraviolet (UV-B) light for 0, 1, 2, 4, 8 hours, respectively.
(3) Colony diameter, spore yield and germination rate
200. Mu.L of the suspension was dropped onto the center of a PDA plate, incubated at 26℃for 10 days, the colony diameter thereof was measured and recorded every day, 100 mL of 0.05% Tween-80 was used for elution after the measurement on the last day was completed, a conidium suspension was prepared, and the conidium yield was calculated by using a hemocytometer.
2mL of the suspension is added into 18mL of SDA culture medium, the mixture is gently mixed and placed into a shaking table at 160 rpm and 25 ℃ for culture, and the germination of spores treated by each treatment is respectively examined by a mirror for 24 hours, 48 hours and 72 hours.
(4) Data processing
SPSS 19.0 software is used for experimental treatment analysis, single-factor analysis of variance is used for analyzing each result, and Tukey is used for checking the difference significance
3. Experimental results
(1) Colony diameter, spore yield
Experimental knotThe results show that after 0, 1, 2, 4 and 8 hours of (UV-B) irradiation, the colony diameters and the spore yields of the aerospace strains SCAUHT18, SCAUHT38 and SCAUHT56 are significantly higher than those of the original strains and other strains after 10 days of growth by comparing the colony diameters of the original strains with those of the aerospace strains SCAUHT18, SCAUHT38 and SCAUHT 56. After 4h of (UV-B) irradiation, the colony diameters of the aerospace strains SCAUHT18, SCAUHT38, SCAUHT56 at 10d were 49.5mm, 45mm, 47mm, while the colony diameters of the original strain HNSB110 were: 31.5mm. The spore yield of the original strain HNSB110 is 1.5X10 at 10d 7 While the space bacterial strain SCAUHT18, SCAUHT38, SCAUHT56 produced spore amount at 10d is respectively: 3.3X10 7 /mL、3.35×10 7 /mL、2.95×10 7 /mL。
The results show that: under the irradiation of different ultraviolet time, the space bacterial strains SCAUHT18, SCAUHT38 and SCAUHT56 have colony diameters (figure 3) and spore yields which are larger than those of the original bacterial strain and other space bacterial strains, have better spore activity, and belong to potential field excellent biocontrol bacteria.
TABLE 2 colony diameter of different strains under UV irradiation at different times
Note that, through Tukey test, different lower case letters in the same column indicate that the difference of colony diameters among different strains is remarkable (P < 0.05)
TABLE 3 1X 10 strains under UV irradiation at different times 6 Spore yield/mL
Note that, through Tukey test, different lower case letters in the same column indicate that the difference of spore production among different strains is remarkable (P < 0.05)
(2) Germination rate
Experimental results show that after different ultraviolet irradiation times, the spore germination rates of the aerospace strains SCAUHT18, SCAUHT38 and SCAUHT56 at 24 hours, 48 hours and 72 hours are higher than those of the original strains and other strains. After ultraviolet irradiation for 4 hours, the germination rate of the aerospace strains SCAUHT18, SCAUHT38 and SCAUHT56 at 24 is 26.5 percent, 24.5 percent and 24 percent, and the germination rate at 48 hours is as follows: 38.5%,36.5% germination rate at 72 h: 74.5%, 72.5%, 70.5%. After the original strain is irradiated for 4 hours by ultraviolet, the germination rate under 24 hours, 48 hours and 72 hours is as follows: 11.5%, 28.5% and 52%.
The results show that the spore germination rates of the aerospace strains SCAUHT18, SCAUHT38 and SCAUHT56 are all larger than those of the original strains and other aerospace strains, and the aerospace strains belong to the potential good field biocontrol bacteria.
TABLE 4 germination Rate of different strains under irradiation with UV 0h
Note that, through Tukey test, different lower case letters in the same column indicate that the germination difference between different strains is remarkable (P < 0.05)
TABLE 5 germination Rate of different strains under irradiation with ultraviolet radiation for 1h
Note that, through Tukey test, different lower case letters in the same column indicate that the germination difference between different strains is remarkable (P < 0.05)
TABLE 6 germination Rate of different strains under irradiation with ultraviolet 2h
Note that, through Tukey test, different lower case letters in the same column indicate that the germination difference between different strains is remarkable (P < 0.05)
TABLE 7 germination Rate of different strains under UV 4h irradiation
Note that, through Tukey test, different lower case letters in the same column indicate that the germination difference between different strains is remarkable (P < 0.05)
TABLE 8 germination Rate of different strains under UV 8h irradiation
Note that, by Tukey test, different lower case letters in the same column indicate that the germination rate difference between different strains is significant (P < 0.05).
Claims (10)
1. An excellent space returning strain with pathogenicity to common thrips is beauveria bassianaBeauveria bassiana) The strain SCAUHT18 has a deposit number of GDMCC NO. 62188.
2. The use of the strain according to claim 1 for controlling common thrips in the field.
3. The use according to claim 2, wherein the strain is cultured to produce spore fluid, spore powder.
4. The use according to claim 3, wherein the beauveria bassiana is administered at a final concentration of 1 x 10 6 ~1×10 9 conidium/mL.
5. The use according to claim 4, wherein the beauveria bassiana is administered at a final concentration of 1 x 10 7 ~1×10 8 conidium/mL.
6. The use according to claim 3, wherein the beauveria bassiana has a final concentration of 10% when administered 8 conidium/mL.
7. The use according to claim 3, wherein the application is performed at the time of predicting that normal thrips will occur or at the beginning of the occurrence.
8. The use according to claim 7, wherein the administration is repeated every 5-7 days.
9. A microbial agent for controlling common thrips, the active ingredient of which is the strain of claim 1.
10. The microbial inoculum of claim 9, which is spore liquid or spore powder.
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