CN115948251B - Wild cordyceps javanica biocontrol strain CJ01 and application thereof - Google Patents

Abstract

A wild cordyceps javanica biocontrol strain CJ01 is separated from bemisia tabaci bodies dying from diseases and is classified and named as cordyceps javanica (Cordyceps javanica) CJ01, and the preservation number is CCTCC NO: m20221721. The strain CJ01 has high-efficiency, specific and stable insecticidal activity on heavy agricultural pests such as bemisia tabaci, aphids and thrips, has low preparation cost and small environmental pollution, is expected to be developed into a microbial pesticide, is used for preventing and controlling the pests caused by the heavy agricultural pests such as bemisia tabaci, aphids and thrips, and meets the important requirements of China on the green sustainable comprehensive prevention and control of the pests.

Description

Wild cordyceps javanica biocontrol strain CJ01 and application thereof

Technical Field

The invention belongs to the fields of microorganism and biological control, and relates to a biocontrol strain CJ01 of Cordyceps javanica (Cordyceps javanica), and high-efficiency insecticidal activity of the strain.

Background

Pests are a serious problem faced in the development process of modern agriculture in all countries of the world. The annual agricultural losses caused by insect pests are hundreds of millions, and the adoption of effective means to reduce the occurrence of insect pests and to increase the yield of crops is an important issue of concern to plant protection workers. The host range of the bemisia tabaci, aphids and thrips which are damaged by the bemisia tabaci, aphids and thrips is wide, and the bemisia tabaci, aphids and thrips can transmit various plant virus diseases, so that serious damage is brought to the growth of crops, huge economic loss is caused, and the bemisia tabaci, aphids and thrips become serious pests which cannot be light in the current agricultural production. For a long time, agricultural workers at home and abroad mainly rely on chemical pesticides for pest control. However, with the abuse and unreasonable application of chemical pesticides, a great deal of natural enemy insects die, the pollution problem caused by pesticide residues to soil, water and other environments is outstanding, and meanwhile, the pesticide resistance of pests is directly caused to a certain extent. Therefore, it has been urgent to explore new environmental friendly non-chemical control means. Compared with the synthetic chemical pesticide, the microbial pesticide developed by natural pathogenic bacteria is used for biological control of target pests, has the characteristics of strong insecticidal specificity, environmental protection, low research and development cost and the like, is an important mode in the field of green pest control, and has great application potential.

In pathogenic microorganisms applied to pest control, the entomogenous fungi have great development potential and application popularization value due to the advantages of various types, complex metabolic types, safety, effectiveness, easy mass production, small environmental pollution and the like, and are the first choice for planting green, safe and high-yield crops. The total number of entomogenous fungi reported worldwide is about 100 and more than 800. Among these entomogenous fungi, two main classes, namely, mycelial fungi (hyphomyces) belonging to the phylum of the subdivision Deuteromycotina, including many kinds that have been widely used, such as Beauveria spp, metarhizium spp, paecilomyces spp, verticillium spp, etc.; second, entomophthora fungi (entomophthlides) belonging to the phylum zygomycota contain many species that often cause high-intensity pest epidemics, such as Entomophthora (Pandora spp.), phytophthora (Erynia spp.), entomophthlora spp, and the like. However, among the published entomopathogenic fungi, only a small number of species resources are developed as fungal pesticides, and the application potential of a large number of existing species is still urgent to be explored, so that the existing available entomopathogenic fungi biocontrol agents are single in species, very deficient in species and limited in pest control range. Meanwhile, the current species resource library further restricts the research and development speed of the biocontrol agent of the entomogenous fungi and the optimization and improvement of the existing preparation, and is extremely unfavorable for the development of the current green agriculture. Therefore, there is a need to continuously separate more effective entomogenous fungi strains from the field to supplement the existing entomogenous fungi resource library, screen and separate new entomogenous fungi with high toxicity, enrich the insecticidal fungi resource library, and provide new species sources for sustainable green prevention and control of crops and commercialization of engineering strains. Therefore, the searching and digging of new available entomogenous fungi resources has very important significance for green agricultural biological control in China.

Disclosure of Invention

The invention aims to provide a wild biocontrol strain-cordyceps javanica (Cordyceps javanica) strain which has high-efficiency insecticidal activity on major agricultural pests such as bemisia tabaci, aphids and thrips and has environment-friendly effect on solving the problems in the prior art.

The wild biocontrol strain is obtained by separating and screening a strain from adult bemisia tabaci parasitica dead on leaves of hibiscus flower, and the strain is identified as cordyceps javanica (Cordyceps javanica), named cordyceps javanica (Cordyceps javanica) CJ01 and preserved in China center for type culture collection (CCTCC NO) at the month 11 of 2022: m20221721, the preservation address is China, university of Wuhan.

The strain CJ01 of the invention has the following characteristics:

1. characteristics of culture

Under the condition of culturing for 10d in a 12L:12D illumination incubator at 28 ℃, the strain CJ01 grows at the fastest speed on an SDA culture medium and grows slowly on a PPDA, SMAY, czapek culture medium; the SDA culture medium has the largest spore yield, and the PDA culture medium has no obvious difference in the spore yields of the two culture media; the strain cultured by SDA has compact and thick colony and good nutrition condition. SDA medium was selected as the optimal medium for the growth of the CJ01 strain. The optimal culture temperature of the strain CJ01 on the SDA culture medium is 28 ℃, the optimal photoperiod is 24L to 0D, and the optimal pH is 6 to 7.

2. Morphological features:

the strain CJ01 has mycelium and velvet shape and is radially grown on an SDA culture medium, the strain is white in early culture period (figure 1A), the strain is off-white in back surface, and mature colonies are light brown (figure 1B). Conidia are oval, circular or oval (fig. 1C), spore-forming cells are thin at their ends and expand at their bases (fig. 1D).

3. Physiological and biochemical characteristics

The strain CJ01 has positive glucose fermentation, lactose fermentation, sucrose utilization, starch hydrolysis, cellobiose hydrolysis, grease hydrolysis and urea test, and the pH value range is 4-10.

The total DNA of the strain CJ01 is extracted, ITS sequence detection is carried out by using universal primers ITS1/ITS4, homology comparison is carried out, and a phylogenetic tree is constructed (figure 2). As can be seen from phylogenetic tree, the strain CJ01 and the Cordyceps javanica Cordyceps javanica belong to the same genus as one genetic branch and have higher support. The strain CJ01 is determined to be the Cordyceps javanica (Cordyceps javanica) and named as the Cordyceps javanica (Cordyceps javanica) CJ01 by combining culture characteristics, morphological characteristics and physiological and biochemical characteristics.

The spore suspension of the strain CJ01 has stronger inhibition effect on tobacco whiteflies, aphids and thrips which are major agricultural pests, and has higher infection insecticidal effect. The spore suspension of the strain CJ01 can be used for preventing and controlling tobacco whiteflies, aphids and thrips which are major agricultural pests. Wherein, the preparation of the spore suspension of the strain CJ01 of the invention is carried out according to the conventional preparation method of spore suspension in the field.

In view of the high infection insecticidal effect of the strain CJ01 of the invention on the heavy agricultural pests such as bemisia tabaci, aphids and thrips, the strain CJ01 can be prepared into a microbial pesticide for preventing and controlling diseases caused by the heavy agricultural pests such as bemisia tabaci, aphids and thrips.

The microbial pesticide takes the strain CJ01 or the spore suspension thereof as an active ingredient, and can be in a solid state form or a liquid state form. The microbial pesticide may also contain other substances with synergistic effect with the strain CJ01. For example, the substance may be a substance derived from a microorganism and inducing disease resistance in plants, a compound inducing disease resistance in plants, other microorganisms inducing disease resistance in plants, or the like; and the substance can be a microorganism source antibacterial substance, can also be an antibacterial compound, can also be other microorganisms with antibacterial effect, and the like.

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

(1) The strain CJ01 of the Cordyceps javanicus is obtained by separating and screening strains from adult bemisia tabaci bodies died by diseases on leaves of hibiscus flowers, and the strain CJ01 is found to have higher infection insecticidal effect on major agricultural pests by measuring toxicity of the strain to the major agricultural pests bemisia tabaci, aphids and thrips.

(2) The strain CJ01 is a biological control strain with important significance in controlling major agricultural pests such as bemisia tabaci, aphids and thrips, can be developed into a microbial pesticide, and provides technical support for green and efficient control of the major agricultural pests such as bemisia tabaci, aphids and thrips.

Drawings

FIG. 1 is a morphology of strain CJ01 of the present invention;

culturing the upper surface of the colony on the 4 th day; B. culturing the upper surface of the colony on the 10 th day; C. conidium; D. and (5) spore-producing structure.

FIG. 2 is a molecular characterization phylogenetic tree of strain CJ01 of the present invention.

Figure 3 is a graph of corrected mortality at day 3 for target pests treated with different spore concentrations.

Detailed Description

EXAMPLE 1 isolation preparation of the Strain CJ01 of the present invention

Isolation of strains: the adult bemisia tabaci bodies which are parasitic to the leaves of the hibiscus flowers and die in illness are sourced from the region of the Hibiscus flower market, the surface stains of the bodies are cleaned by using sterile distilled water, naturally dried, soaked in 75% alcohol for 30-45s for surface disinfection, washed by sterile distilled water for three times, placed on a potato glucose culture medium (PDA), placed at 28 ℃ and 12L:12D and with relative humidity (50+/-5)% for 3-6d, and then the fresh mycelia are picked up around the bodies, transferred to a PDA inclined plane for separation culture, transferred to the PDA culture medium for 10d after colony formation, and picked up into 1X 10 after the conidia are formed ⁶ spore/mL conidium suspension, 100 μl of suspension was aspirated and smeared evenly onto new PDA for further culture for 10d; purifying and culturing for multiple times to obtain single bacterial colonies, and respectively storing the single bacterial colonies for later use.

Screening of strains: the adoption of high-yield high-quality strain is a primary premise for obtaining better control effect. Therefore, in screening for superior isolates, the spore yield, pathogenicity and colony growth rate of the strain are important reference indexes. After objectively comparing the three indexes of each isolated strain, the strain CJ01 is found to have good performance in terms of growth speed, spore yield and pathogenicity to bemisia tabaci adults, and meets various standards of high-yield and high-quality strains.

Example 2 indoor pathogenicity test 1

1. Test insect source: bemisia tabaci;

2. the test method comprises the following steps: the bioassay method uniformly adopts a spray method;

3. preparation of spore suspension:

the slant test tube containing the strain CJ01 of the invention is inoculated on SDA culture medium, activated in a constant temperature incubator with optimal temperature and optimal photoperiod, and transferred to a new SDA plate for culture for 10d. After mass production, a small amount of sterilized 0.1% Tween-80 sterile aqueous solution was added, and the mycelia were scraped with a spatula to collect conidia. Pouring the bacterial liquid into a sterilizing beaker, scattering accumulated spores to enable the spores to be uniformly suspended, and filtering the spores by using 4 layers of sterilizing medical gauze. Estimating the resulting spore concentration using a hemocytometerDegree, it is formulated to a concentration of 1X 10 ⁵ 、1×10 ⁶ 、1×10 ⁷ And 1X 10 ⁸ spores/mL four gradients of suspension at the concentration to be tested.

4. Insecticidal test

And respectively spraying the spore suspension liquid with the concentration to be tested on leaf surfaces of the eggplant leaves after the sterilization treatment, airing the leaves, and then inoculating 100 bemisia tabaci adults, wherein each treatment is repeated for 3 times. The experimental plants were placed in a constant temperature climatic chamber at 28+ -1deg.C, 12L:12D, and kept at a humidity above 50%, and the number of dead insects and the calculated mortality were recorded daily for a total of 3 days. The results show that mortality of bemisia tabaci increases with increasing spore suspension concentration and time, 1×10 at different suspension concentrations ⁸ The spore/mL treatment concentration was the best at day 3 infestation insecticidal (see also figure 3). At 1X 10 ⁸ LC of the strain CJ01 on Bemisia tabaci at 3 rd day at spore/mL concentration ₅₀ The value was 4.33X10 ⁶ spores/mL (2.59X10) ⁶ -7.49×10 ⁶ spores/mL), see table 1 below.

TABLE 1 determination of the biological Activity of the Strain CJ01 of the invention against Bemisia tabaci

	Intercept of (intercept of)	Slope of	Chi square value	df	LC 50 (95% confidence interval) (spore/mL)
						Bemisia tabaci (Bemisia tabaci)	-0.46	0.82	0.94	2	4.33×10 6 (2.59×10 6 -7.49×10 6 )

Example 3 indoor pathogenicity test two

1. Test insect source: aphids and thrips;

2. the test method comprises the following steps: the bioassay method uniformly adopts a spray method;

3. the preparation of spore suspensions is described in example 2;

4. the procedure of the insecticidal test is described in example 2;

the results showed that mortality of aphids and thrips increased with increasing spore suspension concentration and time, treatments with different spore concentrations showed the highest insecticidal effect on day 3, 1×10 ⁸ The spore/mL treatment concentration showed the best infestation insecticidal effect on day 3 (see also figure 3). LC of the strain CJ01 of the invention against aphids and thrips on day 3 ₅₀ The values are respectively 1.05X10 ⁷ spores/mL (5.71×10) ⁶ -2.39×10 ⁷ spores/mL) and 9.65X10 ⁶ spores/mL (5.32X10) ⁶ -2.05×10 ⁷ spores/mL), see table 2 below.

TABLE 2 determination of the biological Activity of the Strain CJ01 of the invention against aphids and thrips

	Intercept of (intercept of)	Slope of	Chi square value	df	LC 50 (95% confidence interval) (spore/mL)
						Thrips horse	-0.76	0.82	4.16	2	1.05×10 7 (5.71×10 6 -2.39×10 7 )
Aphids	0.74	0.61	3.30	2	9.65×10 6 (5.32×10 6 -2.05×10 7 )

In conclusion, the cordyceps javanica (Cordyceps javanica) CJ01 strain provided by the invention has a strong inhibition effect on tobacco whiteflies, aphids and thrips which are major agricultural pests, has a high infection insecticidal effect, is environment-friendly, and does not cause secondary pollution to the ecological environment. The cordyceps javanica (Cordyceps javanica) CJ01 is suitable for biological control of tobacco whiteflies, aphids and thrips which are major agricultural pests, and is a supplement to a entomogenous fungi resource library under the condition that wild entomogenous fungi strains available at the present stage are deficient.

Claims (10)

1. Cordyceps javanica (Cordyceps javanica) CJ01 with preservation number of CCTCC NO: m20221721.

2. Use of cordyceps javanica CJ01 according to claim 1 for controlling bemisia tabaci.

3. Use of cordyceps javanica CJ01 according to claim 1 for controlling aphids.

4. Use of cordyceps javanica CJ01 according to claim 1 for controlling cynara scolymus.

5. The spore suspension of Cordyceps javanica CJ01 of claim 1.

6. Use of the spore suspension of claim 5 for controlling bemisia tabaci.

7. Use of a spore suspension as defined in claim 5 for controlling aphids.

8. Use of a spore suspension as defined in claim 5 for controlling thistle.

9. A microbial pesticide comprising the Cordyceps javanica CJ01 of claim 1 or the spore suspension of claim 5.

10. Use of a microbial insecticide according to claim 9 for controlling bemisia tabaci, aphids and/or thrips.