Cordyceps javanica PYZK43 with prevention and control effect on diaphorina citri and application of cordyceps javanica PYZK43

Abstract

The invention discloses a cordyceps javanica strain PYZK43 with prevention and control effects on diaphorina citri and application of the cordyceps javanica strain PYZK43. The cordyceps javanica strain is named as cordyceps javanica PYZK43, and the preservation number of the cordyceps javanica strain is GDMCC No: 65819. The cordyceps javanica strain is named as cordyceps javanica PYZK43. The strain has a very strong diaphorina citri killing effect, after diaphorina citri adults are inoculated at two spore concentrations of 1 * 10 < 7 > spores / mL and 1 * 10 < 8 > spores / mL for 9 days, the death rate of the adults reaches 100%, the insecticidal effect is superior to that of the existing known same strain, and the strain has very strong application potential in biological control of diaphorina citri.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological control, and particularly relates to a Cordyceps javanica strain PYZK43 with a control effect on Diaphorina citri and its application. Background Art

[0002] Diaphorina citri is an important pest of citrus crops. It not only directly harms the new shoots and young leaves of citrus, but also is the natural vector for transmitting Citrus Huanglongbing (HLB). Citrus Huanglongbing is known as the "cancer of citrus", causing devastating blows to the citrus industry, leading to decreased yields, deteriorated fruit quality, and huge economic losses. At present, the control of Diaphorina citri mainly relies on chemical pesticides. However, the long-term use of chemical pesticides has led to a series of problems, including environmental pollution, pesticide residues, enhanced pest resistance, and harm to non-target organisms. Therefore, the development of efficient, low-toxic, and environmentally friendly biological control methods has become the focus of current research.

[0003] Biological control technology uses natural enemy insects, microorganisms, or their metabolites to control pests, and has the advantages of high efficiency, low toxicity, low residue, and low resistance. In the biological control of Diaphorina citri, entomopathogenic fungi (such as Paecilomyces lilacinus, Verticillium lecanii, Cladosporium cladosporioides, etc.) have been proven to have significant pathogenic effects.

[0004] In recent years, the research on Cordyceps javanica in the field of biological control has gradually increased. For example, South China Agricultural University screened a Cordyceps javanica strain GZQ-1 with high pathogenicity, which showed strong infectivity to Bemisia tabaci and Diaphorina citri, and could be applied to the control of diseases caused by pathogens transmitted by Bemisia tabaci and Diaphorina citri. However, the mortality rate of adult Diaphorina citri infected with the Cordyceps javanica strain GZQ-1 at a concentration of 1×10 7 spores / mL spore suspension for 7 days was 72.2±2.94%, and the control effect needs to be improved.

[0005] In summary, there are still few studies on Cordyceps javanica strains against Diaphorina citri at present. In particular, the screening of highly efficient strains, the study of pathogenic mechanisms, and the evaluation of their effects in field applications still need to be further explored. Summary of the Invention

[0006] The first object of the present invention is to provide a Cordyceps javanica strain PYZK43 for controlling Diaphorina citri, and its preservation number is: GDMCC No: 65819.

[0007] The second object of the present invention is to provide the application of the above-mentioned Cordyceps javanica strain PYZK43 in controlling Diaphorina citri.

[0008] The third object of the present invention is to provide the application of the above-mentioned Cordyceps javanica PYZK43 in the preparation of a biological pesticide for controlling Diaphorina citri

[0009] The fourth object of the present invention is to provide the application of the above-mentioned Cordyceps javanica PYZK43 in controlling plant diseases caused by Diaphorina citri

[0010] The fifth object of the present invention is to provide the application of the above-mentioned Cordyceps javanica PYZK43 in the preparation of a biological pesticide for controlling plant diseases caused by Diaphorina citri

[0011] Preferably, the disease is citrus huanglongbing

[0012] The sixth object of the present invention is to provide an agent for controlling Diaphorina citri, which comprises the above-mentioned Cordyceps javanica PYZK43

[0013] The seventh object of the present invention is to provide an agent for controlling citrus huanglongbing, which comprises the above-mentioned Cordyceps javanica PYZK43

[0014] Preferably, the agent is a bacterial suspension of Cordyceps javanica PYZK43, with a concentration of 1×10 4 ~10 8 spores / mL

[0015] The eighth object of the present invention is to provide a method for controlling Diaphorina citri, which comprises the step of contacting the living bacteria of the above-mentioned Cordyceps javanica PYZK43 or a culture containing the living bacteria of the above-mentioned Cordyceps javanica PYZK43 with the body of Diaphorina citri

[0016] The present invention has the following beneficial effects:

[0017] The present invention has screened out a strain of Cordyceps javanica PYZK43 with high efficiency in controlling Diaphorina citri. After inoculating adult Diaphorina citri with 2 spore concentrations of 1×10 7 spores / mL and 1×10 8 spores / mL for 9 days, the mortality rate of adults reached 100% in both cases. The insecticidal effect is better than that of the existing known strains of the same species, and it has great application potential in the biological control of Diaphorina citri

[0018] Cordyceps javanica PYZK43 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on January 17, 2025, with the deposit number GDMCC No: 65819, and the deposit address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Postcode: 510070 Description of the Drawings

[0019] Figure 1 The lethal effects of Cordyceps javanica strain PYZK43 at different concentrations on the adults of Diaphorina citri Detailed implementation manners

[0020] The following examples are further illustrations of the present invention rather than limitations thereof.

[0021] Example 1: Isolation and identification of the strain

[0022] 1. Isolation and purification of pathogenic fungi

[0023] In May - June 2024, the mummified Diaphorina citri were collected from Murraya paniculata in Panyu District, Guangzhou. The mummified Diaphorina citri collected in the field were placed in 1.5 mL centrifuge tubes and taken back to the laboratory. They were observed and photographed under a stereomicroscope and numbered respectively. Referring to the method of isolation and purification by Pu Zhelong et al. (1996), the treatment was carried out on a super - clean workbench: when no visible mycelium grew outside the body wall of the mummified insect, the insect cadaver was first disinfected with 70% absolute ethanol for 30 s, then placed in 1% sodium hypochlorite solution for about 1 min, and finally washed 3 times with sterile ultrapure water, about 45 s each time. After sucking the excess water on the surface of the insect body with sterile filter paper, each insect cadaver was transferred to PDA medium with small tweezers; when a large amount of mycelium and conidia had formed on the body wall of the mummified insect, the mycelium was directly shaken onto the medium or picked with an inoculation loop for streak isolation. It was cultured in an incubator at a constant temperature of 26 °C.

[0024] The colonies were observed daily. According to the morphological characteristics such as the color, size, and shape of the colonies, different types of colonies on the surface of the medium were picked and placed on a new PDA medium for purification. When the colonies grew to be visible to the naked eye without other colonies, a 6 mm × 6 mm punch was used to take the agar plug at the edge of the colony and put it into a cryotube containing 15% glycerol, and then placed in a - 80 °C refrigerator. A total of 15 strains of pathogenic fungi were obtained according to the above method.

[0025] 2. Identification of pathogenic fungi

[0026] After culturing the 15 strains on PDA plates for 10 d, the mycelium and conidia were scraped with a sterilized pipette tip and placed in a small mortar. Liquid nitrogen was added and ground into powder, which was then loaded into a 1.5 mL centrifuge tube. DNA was extracted according to the fungal DNA extraction kit. Three genes, ITS, TEF, and TUB2 (primer sequences are shown in Table 1), were used for PCR amplification of the 15 strains of pathogenic fungi. After the PCR products were detected by gel electrophoresis, they were sent to Tianyi Company for sequencing.

[0027] Table 1 PCR primers

[0028]

[0029] 3. Results

[0030] The sequences returned by sequencing were compared one by one in NCBI Blast, and it was found that the sequences of 15 strains had 100% similarity with the sequences of multiple strains of Cordyceps javanica in GenBank.

[0031] Example 2: Determination of the pathogenicity of the pathogen of Diaphorina citri

[0032] 1. Preparation of spore suspension

[0033] The mycelial cakes stored in 15% glycerol were inoculated onto PDA plates and cultured in a constant temperature incubator at 26°C for 10 days. The mycelia and conidia on the plates were washed off with 0.05% Tween-80 sterile water, stirred with a magnetic stirrer at room temperature for 20 min, and shaken vigorously to disperse the conidia. The mycelia and impurities were filtered through four layers of degreased cotton gauze. 10 μL of the spore suspension was pipetted onto one side of a hemocytometer (25×16 type), covered with a cover glass, and left to stand for 10 min. The spore suspension concentration was calculated by averaging the counts taken 3 times under a microscope. The spore suspension was serially diluted with 0.05% Tween-80 sterile water to 1×10 8 spores / mL, and stored in a 4°C refrigerator for later use.

[0034] 2. Pathogenicity determination device

[0035] To ensure that the Diaphorina citri had sufficient food sources and that the Murraya exotica survived during the experiment, branches of Murraya exotica with relatively consistent growth conditions were placed in a 25 mm×25 mm weighing bottle containing 6 mL of water, sealed with Parafilm to fix the Murraya exotica as the food source for the Diaphorina citri, and a cylindrical paper tube made of a 9 cm disposable plate, filter paper, and 1 / 3 of an A4 paper was used as the transfer container for the food source, and the food source was placed inside. It was cultured in an artificial climate incubator at 27°C (L:D = 14:10 h, RH: 90% ± 1).

[0036] 3. Screening of pathogenic bacteria

[0037] Healthy adult Diaphorina citri were placed in small mesh bags made of degreased cotton medical gauze, immersed in each concentration of spore suspension for 20 s, excess water was blotted off with a sterilized filter paper, and the still freely movable Diaphorina citri were picked up with a small brush and placed into the device; treatment with 0.05% Tween-80 sterile water was used as the control group, and then it was cultured in an artificial climate incubator at 27 ± 1°C (L:D = 14:10 h, RH: 90% ± 1). The cumulative number of dead Diaphorina citri was observed every 24 h, and a Diaphorina citri was considered dead when it lay on its side, flipped over, and did not move when gently touched with a brush. Each bacterium was treated with 1 concentration, with 3 biological replicates, and each replicate had 30 adult Diaphorina citri.

[0038] 4. Results

[0039] (1) Pathogenicity of each strain to Diaphorina citri

[0040] Through the above methods of isolation, purification and pathogenicity determination, a total of 14 strains that met Koch's postulates were screened out and named PYZK01, PYZK31, PYZK32, PYZK33, PYZK36, PYZK37, PYZK39, PYZK41, PYZK43, PYZK44, PYZK45, PYZK46, PYZK47, and PYZK49 respectively. When these strains were used to treat adult Diaphorina citri, the cumulative mortality of adults on the 10th day is shown in Table 2.

[0041] Table 2 Pathogenicity of each strain of Cordyceps javanica to adult Diaphorina citri

[0042]

[0043] Note: Different lowercase letters indicate significant differences in each column index among different treatments.

[0044] As can be seen from Table 2, each strain of Cordyceps javanica has pathogenicity to adult Diaphorina citri. Among them, the strain PYZK43 has the highest cumulative mortality rate for adult Diaphorina citri. Therefore, the PYZK43 strain was selected for subsequent experiments. The PYZK43 strain was named Cordyceps javanica PYZK43, which was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on January 17, 2025. The deposit number is GDMCC No: 65819, and the deposit address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Postcode: 510070.

[0045] Example 3: Lethal effect of Cordyceps javanica PYZK43 strain with different concentrations on adult Diaphorina citri

[0046] Referring to the method of Example 2, the lethal effects of inoculating adult Diaphorina citri with spore suspensions of Cordyceps javanica PYZK43 strain at different concentrations (1×10 4 spores / mL, 1×10 5 spores / mL, 1×10 6 spores / mL, 1×10 7 spores / mL, 1×10 8 spores / mL) at different times were evaluated. The results are shown in Figure 1 .

[0047] As Figure 1 shown, after inoculating adult Diaphorina citri with conidial suspensions of Cordyceps javanica at different concentrations for 10 days, the cumulative mortality of adults increased with the passage of inoculation time and the increase of spore dose. 1×107 spores / mL and 1×10 8 After inoculating adult Diaphorina citri Kuwayama with two spore concentrations of 1×10 4 spores / mL and 1×10 8 spores / mL for 7 days, the adult mortality rates were (88.89±1.92)% and (97.78±1.92)% respectively. After inoculating adult Diaphorina citri Kuwayama for 8 days, the adult mortality rates were (95.56±1.93)% and (100.00±0.00)% respectively. After inoculating for 9 days, the adult mortality rate reached 100%. The cumulative mortality rate of the lowest concentration of 1×10

[0048] Example 4: Lethal time of Cordyceps javanica (Teng) Kob. strain PYZK43 to adult Diaphorina citri Kuwayama

[0049] Referring to the method of Example 2, evaluate the lethal time of inoculating adult Diaphorina citri Kuwayama with spore suspensions of Cordyceps javanica (Teng) Kob. strain PYZK43 at different concentrations (1×10 4 spores / mL, 1×10 5 spores / mL, 1×10 6 spores / mL, 1×10 7 spores / mL, 1×10 8 spores / mL). The results are shown in Table 3.

[0050] Table 3 Lethal time of Cordyceps javanica (Teng) Kob. strain PYZK43 to adult Diaphorina citri Kuwayama

[0051]

[0052]

[0053] As shown in Table 3, after inoculating adult Diaphorina citri Kuwayama with spore suspensions of Cordyceps javanica (Teng) Kob. strain PYZK43 prepared at different concentrations, its LT 50 and LT 95 gradually increased with the decrease of spore concentration. 1×10 8 spores / mL, 1×10 7 spores / mL, 1×10 6 spores / mL, 1×10 5 spores / mL and 1×10 4LT at spores / mL 50 were 4.17 d, 5.02 d, 6.56 d, 8.58 d, and 11.13 d, respectively, and LT 95 were 6.41 d, 8.18 d, 14 d, 19.17 d, and 27.91 d, respectively.

[0054] Example 5: Lethal concentration of Cordyceps javanica strain PYZK43 against adult Diaphorina citri

[0055] Referring to the method of Example 2, adult Diaphorina citri were inoculated with spore suspensions of Cordyceps javanica strain PYZK43 at different concentrations. On the 4th, 5th, 6th, 7th, 8th, 9th, and 10th days after inoculation, the death of adult Diaphorina citri was observed and recorded respectively, and the LC 50 (median lethal concentration) and LC 95 (95% lethal concentration) at different time points were calculated and statistically analyzed to obtain the virulence regression equation and the correlation coefficient R. The results are shown in Table 4.

[0056] Table 4 Lethal concentration of Cordyceps javanica strain PYZK43 against adult Diaphorina citri

[0057]

[0058]

[0059] As can be seen from Table 4, with the increase of inoculation time, the concentration of conidia required to infect adult Diaphorina citri gradually decreased. The LC 50 were 1.19×10 8 , 1.50×10 7 , 1.31×10 6 , 2.73×10 5 , 1.30×10 5 , 5.10×10 4 , and 2.95×10 4 spores / mL at 4, 5, 6, 7, 8, 9, and 10 d after inoculation, respectively, and the LC 95 were 2.02×10 11 , 3.30×10 10 , 1.11×10 9 , 8.33×10 7 , 3.92×10 7 , 5.97×10 6 , 5.13×10 6 spores / mL, respectively.

[0060] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the protection scope of the present invention should be defined by the scope of the claims. For those of ordinary skill in the art, without departing from the spirit and scope of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as within the protection scope of the present invention.

Claims

1. Cordyceps javanica PYZK43, whose deposit number is: GDMCC No: 65819.

2. Use of the Cordyceps javanica PYZK43 described in claim 1 in controlling citrus psyllids.

3. Use of the Cordyceps javanica PYZK43 according to claim 1 in the preparation of a biological pesticide for controlling citrus psyllids.

4. Use of the Cordyceps javanica PYZK43 according to claim 1 in preventing and controlling plant diseases caused by citrus psyllid.

5. Use of the Cordyceps javanica PYZK43 according to claim 1 in the preparation of a biological pesticide for preventing and controlling plant diseases caused by citrus psyllid.

6. The use according to claim 4 or 5, characterized in that: The disease is citrus Huanglongbing.

7. A medicament for controlling citrus psyllids, characterized in that: The invention comprises the Javanese Cordyceps sinensis PYZK43 as claimed in claim 1.

8. A medicament for preventing and treating citrus Huanglongbing disease, characterized in that: The invention comprises the Javanese Cordyceps sinensis PYZK43 as claimed in claim 1.

9. The medicament according to claim 7 or 8, characterized in that The agent is a suspension of Cordyceps javanica PYZK43, with a concentration of 1×10 4 ~10 8 spores / mL.

10. A method for controlling citrus psyllids, characterized in that: The method comprises the step of contacting the live bacteria of the Cordyceps javanica PYZK43 according to claim 1 or a culture containing the live bacteria of the Cordyceps javanica PYZK43 according to claim 1 with citrus psyllid bodies.

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