Alternania iridiaustrialis JY-4 and application thereof in prevention and treatment of pomelo karaoke

Abstract

The invention discloses an Alternania iridiaustrialis JY-4 strain and an application of the Alternania iridiaustrialis JY-4 strain in prevention and treatment of Kallkala pomelo, and belongs to the technical field of microbial prevention and treatment of plant insect pests. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 11, 2025, the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.42162. According to the invention, an entomogenous fungus separated from a stiff aphid body is comprehensively judged through indoor toxicity determination and field control effect tests, and the alternaria alternata JY-4 has a control effect on the adult Kallkala and provides a theoretical support for comprehensive treatment and scientific prevention and control of Kallkala.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial control of plant pests, and particularly relates to a strain of Alternaria iridiaustralis JY-4 and application thereof in preventing and treating Diaphorina citri. BACKGROUND

[0002] Citrus Citrus reticulata Blanco, a woody plant of Rutaceae, has the largest planting area and yield in the world since the 21st century, and plays an indispensable role in promoting farmers' income. Citrus is the fruit tree with the widest planting area and the highest economic weight in southern China. Thanks to the diverse and rich climate types, water resources and soil nutrients in Yunnan, the planting scale of citrus ranks second in the province's fruit industry.

[0003] However, with the improvement of citrus industry benefits and the expansion of planting area, citrus diseases and pests are becoming more and more serious, and citrus production has been seriously affected. Diaphorina citri Cacopsylla citrisuga Diaphorina citri belongs to Hemiptera Psyllidae. Diaphorina citri mainly occurs in citrus-producing areas above 1000 m above sea level, and the larval population of Diaphorina citri is most serious in lemon-producing areas at an altitude of 1000-1500 m. The occurrence dynamics of the population number shows a significant positive correlation with lemon flowering and altitude. The higher the altitude, the more serious the damage.

[0004] Due to the multiple annual occurrence of Diaphorina citri in citrus orchards, high concealment, and rich citrus varieties, it is very difficult to control Diaphorina citri, and the ecological problems caused by it are becoming more and more serious. The main means of controlling Diaphorina citri in the field is chemical pesticide control, but due to the unscientific use method and the lack of attention to biological natural enemies, it not only has poor control effect, but also has great negative impact. SUMMARY

[0005] The purpose of the present application is to provide a strain of Alternaria iridiaustralis JY-4 and application thereof in preventing and treating Diaphorina citri, so as to solve the problems in the prior art.

[0006] To achieve the above purpose, the present application provides the following solutions. One of the technical solutions of the present application is a strain of Alternaria iridiaustralis JY-4, which was preserved in the China General Microbiological Culture Collection Center on August 11, 2025, and the address is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 42162.

[0007] The second technical solution of the present application is a microbial inoculant, which comprises theAlternaria iridiaustralis JY-4.

[0008] The third technical solution of the present invention, the Alternaria iridiaustralis Application of JY-4 or the aforementioned microbial agent in the preparation of plant pest control for the control of the teak psyllid.

[0009] The fourth technical solution of this invention is a method for preventing and controlling plant pests caused by the psyllid, utilizing the aforementioned... Alternaria iridiaustralis The plants are treated by spraying JY-4 or the aforementioned microbial agent.

[0010] The fifth technical solution of this invention is a product for preventing and controlling plant pests caused by the psyllid, comprising the aforementioned... Alternaria iridiaustralis JY-4 or the aforementioned microbial agent.

[0011] Based on the above technical solution, the present invention has the following technical effects: The entomopathogenic fungus isolated from the body of the aphid in this invention was identified by morphology and molecular biology as Alternaria alternata, JY-4. Alternaria iridiaustralis Based on a comprehensive assessment of both indoor toxicity testing and field efficacy trials, Alternaria JY-4 demonstrated control efficacy against adult citrus psyllids, providing theoretical support for the integrated management and scientific control of citrus psyllids. Attached Figure Description

[0012] Alternaria iridiaustralis Infecting adult psyllids of the grapefruit with JY-4 (1×10⁻⁶) 12 Symptoms (conidia / mL) 14 days later.

[0013] Figure 1 Phylogenetic tree of JY-4 strain based on ITS sequence.

[0014] Figure 2 The image shows the morphology of fungal strain JY-4 on PDA medium. (A) Front view of colony at 5 days; (B) Back view of colony at 5 days; (C) Mycelium of strain JY-4; (D) Conidia of strain JY-4.

[0015] Figure 3 Different concentrations of Alternaria japonica JY-4 spore suspensions (1×10⁻⁶) 1 1×10 3 1×10 6 1×10 9 1×10 12 Indoor toxicity of conidia / mL against adult psyllids.

[0016] Figure 4 Different concentrations of Alternaria japonica JY-4 spore suspensions (1×10⁻⁶) 1 1×10 31 x 10 6 1 x 10 9 1 x 10 12 conidia / mL) on the field mortality of adult of H. aurantii. DETAILED DESCRIPTION

[0017] The technical solutions described in the present application are conventional solutions in the art if not specifically stated, and the reagents or raw materials used are purchased from commercial channels or are already disclosed if not specifically stated.

[0018] The data obtained in the present application are statistically analyzed using Excel 2010, processed and analyzed using SPSS 20.0 statistical software, and plotted using Origin 2021.

[0019] The virulence study data are analyzed using the Probit probability value analysis method to obtain the virulence regression equation, the median lethal concentration LC 50 , the median lethal time LT 50 , the 95% confidence interval, the chi-square value, and the correlation coefficient. The corrected mortality rate formula is: Cumulative mortality rate = (total number of dead insects in the sample / total number of insects in the sample) x 100%; Cumulative corrected mortality rate = (treatment group mortality rate - control group mortality rate) / (1 - control group mortality rate) x 100%.

[0020] The present application provides a strain of Figure 5 JY-4, which was deposited with the China General Microbiological Culture Collection Center on August 11, 2025, at the address of No. 1, Beichen West Road, Haidian District, Beijing, with the preservation number of CGMCC NO. 42162.

[0021] The present application also provides a microbial agent comprising the Alternaria iridiaustralis JY-4.

[0022] The present application also provides the use of the Alternaria iridiaustralis JY-4 or the microbial agent in the preparation of a plant pest control agent for preventing and treating plant pests caused by H. aurantii.

[0023] In some specific embodiments, the plant comprises citrus.

[0024] The present application also provides a method for preventing and treating plant pests caused by H. aurantii, which comprises spraying the plant with the Alternaria iridiaustralis JY-4 or the microbial agent.

[0025] In some specific embodiments, the plant comprises citrus.

[0026] The application also provides a product for preventing and treating plant insect pests caused by Toxoptera citricola, comprising the microorganism agent Alternaria iridiaustralis JY-4 or the microorganism agent.

[0027] In some specific embodiments, Alternaria iridiaustralis The spore concentration of JY-4 is 1×10 1 ~ 1×10 12 conidia / mL.

[0028] Embodiment 1 1 Test material Sample: wild entomogenous fungi parasitizing on the bodies of dead aphids on the tender shoots of citrus, collected in the orange garden of the Ruli Station of the Institute of Tropical and Subtropical Economic Crops, Yunnan Academy of Agricultural Sciences.

[0029] Culture medium: potato dextrose agar medium (PDA).

[0030] 2 Isolation and purification of the strain The diseased dead aphids (cadavers) were immersed in 75% alcohol for 3 s, washed with sterile water for 3 times, then inoculated on a PDA medium plate containing 15% sodium chloride, and cultured in a constant temperature incubator at 25 ± 1 ℃. The culture was observed once a day after inoculation, and the newly generated mycelium around the insect body was picked for isolation and purification until pure colonies were obtained. The pure colonies obtained by isolation were screened by the spraying method to obtain a strain with pathogenicity to Toxoptera citricola, which was named JY-4.

[0031] After being infected with the spore suspension of the JY-4 strain for 14 days in an artificial climate chamber at T=20 ± 1 ℃, RH=80 ± 5%, E=12 000 lx, and L:D=14 h:10 h, the adult bodies of Toxoptera citricola were wrapped with white mycelium. The white mycelium on the bodies of Toxoptera citricola after infection with the spore suspension of the JY-4 strain was relatively slender and not clustered. Alternaria iridiaustralis ).

[0032] 3 Identification of the JY-4 strain (1) Morphological identification of the JY-4 strain The completely purified mycelium was inoculated on new PDA medium and cultured in a constant temperature incubator at 25 ± 1 ℃ for 7 days. The morphology, color, size and other characteristics of the front and back of the colonies were observed every other day. After the strain was cultured to the mature spore production stage, the mycelium of the strain was prepared into a slide and observed under an optical microscope to record the morphology of the mycelium and the conidium.

[0033] The strain obtained after separation and purification is JY-4. After the strain JY-4 is cultured on PDA for 5 days, the colony presents concentric circles, the innermost layer on the front side is gray-brown colony, the second layer is yellow-white colony, and the outermost layer is white colony, and the mycelium presents relatively tight flocculation; the back side of the colony has the same color as the front side of the colony Figure 1 (central A, B); the conidia of the strain JY-4 present inverted rod shape, yellow-brown, with longitudinal and transverse septum, and the mycelium has no septum Figure 3 (central C, D).

[0034] (2) Molecular biological identification of the JY-4 strain The genomic DNA of the fungus is extracted by using a kit (produced by Hangzhou Beiwo Medical Technology Co., Ltd.), 25 uL of an amplification system is used for PCR amplification by using the universal primers ITS1 and ITS4 for fungi. After the PCR product is detected by 1% agarose gel electrophoresis and it is confirmed that the band size is correct, the sample with successful amplification is sent to Wuhan Qikexin Biological Technology Co., Ltd. for sequencing, and the sequencing results are compared by using the BLAST comparison of the NCBI database. The sequence is analyzed by using the MEGA software, and a phylogenetic tree is constructed.

[0035] The biological identification result of the pathogenic strain of the cotton aphid shows that the strain JY-4 is clustered with the strain of Alternaria, and the similarity with Figure 3 (NR 136120.1) is 100%, and the genetic distance with the outgroup is far Alternaria iridiaustralis . In combination with the morphological identification of the strain JY-4, it can be determined that the strain JY-4 is a fungus of the genus Alternaria Figure 2 Alternaria .

[0036] iridiaustralis The JY-4 is preserved in the China General Microbiological Culture Collection Center on August 11, 2025, the address is No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 42162.

[0037] Example 2 Study on the pathogenicity of the JY-4 strain to adult of Diaphorina citri 1 Test material The test insect: Diaphorina citri is collected in a citrus orchard without spraying in Mengxiu County, Dehong Prefecture, Yunnan Province, and is bred and fed for multiple generations in a rearing room at 20°C, RH = 80% ± 5%, L:D = 14 h:10 h by using lemon seedlings.

[0038] The test strain: the JY-4 strain is isolated from diseased citrus aphids.

[0039] 2 Test method (1) Preparation of spore suspension The JY-4 strain was inoculated onto PDA medium and cultured for 10 days in an incubator at (26 ± 2) ℃, L:D = 14 h:10 h, and 3000 lx illumination. Then, well-grown colonies were selected and treated with 0.05% ( The spores were diluted with Tween-80 sterile water, vortexed for 10 minutes, and then filtered to obtain a relatively pure spore suspension. The spore concentration of the mother liquor was determined by counting spores under a 40x microscope using a hemocytometer, and the solution was prepared to a concentration of 1×10⁻⁶ spores. 1 1×10 3 1×10 6 1×10 9 1×10 12 A spore suspension of conidia / mL.

[0040] (2) Determination of the control efficacy of spore suspension against adult psyllids. Clean, pest-free citrus shoots (15-20 cm) were selected, washed, and air-dried. The base of the shoots was wrapped with cotton balls and placed in tissue culture bottles. Thirty adult *Pteris vittata* spores were inoculated into each bottle. Different concentrations of spore suspension were sprayed using a handheld sprayer until the leaves were fully moistened but without forming droplets. The bottle opening was sealed with a 120-mesh screen and secured with a rubber band. The bottles were then placed in an artificial climate chamber (T=20 ± 1 ℃, RH=80 ± 5%, E=12000 lx, L:D=14 h:10 h) for cultivation. The control (CK) was 0.05% (… The same spore concentration and control were tested in Tween-80 sterile water, and the experiment was repeated three times. The number of dead adult spore mites was recorded daily for 7 days or more.

[0041] Under indoor conditions, the pathogenicity of Alternaria JY-4 spore suspensions at different concentrations against adult *Alternaria gracilis* psyllids after 7 days showed that the corrected mortality rate of adult *Alternaria gracilis* psyllids increased with increasing concentration of *Alternaria gracilis* JY-4 spore suspension. Alternaria iridiaustralis ).

[0042] 1×10 12 Treatment with Alternaria conidia / mL Alternaria JY-4 spore suspension for 7 days resulted in a mortality rate of 50.00% against adult psyllids, significantly higher than other concentrations. P<0.05 ), respectively compared to 1×10 9 conidia / mL, 1×10 6 conidia / mL, 1×10 3 conidia / mL, 1×10 1The LC50 concentrations of Alternaria JY-4 spore suspensions after infection were 8.11%, 20.27%, 33.79%, and 41.89%, respectively. Table 1 shows that 7 days after spraying the spore suspension, the LC50 of different concentrations of Alternaria JY-4 spore suspensions against adult *Alternaria cirrhosa* psyllids were significantly higher. 50 They are 7.89×10 7 conidia / mL. 1×10 12 A spore suspension of Alternaria JY-4 at a concentration of conidia / mL showed a LT (LT) effect on adult *Alternaria gracilis* psyllids. 50 It is 6.11 days (Table 2).

[0043] Table 1. Regression equation for the pathogenicity of Alternaria JY-4 to adult citrus psyllids.

[0044] Table 2 1×10 12 Lethal time of Alternaria JY-4 (conidia / mL) against adult grapefruit psyllids

[0045] Example 3 1. Experimental Materials and Methods 1.1 Test Materials Test strain: Alternaria japonica JY-4 was isolated from diseased citrus aphids.

[0046] Potted plants used for testing: lemon seedlings, variety Yun Ning No. 1.

[0047] 1.2 Test Methods The experiment was conducted in a lemon seedling greenhouse at the Ruili Station of the Tropical and Subtropical Economic Crops Research Institute in Dehong Prefecture, Yunnan Province. Well-grown Alternaria JY-4 colonies were selected and treated with 0.05% ( The spores were diluted with Tween-80 sterile water, vortexed for 10 minutes, and then filtered to obtain a spore suspension. The spore concentration of the mother liquor was determined by counting spores under a 40x microscope using a hemocytometer, and prepared into a 1×10⁻⁶ solution. 1 1×10 3 1×10 6 1×10 9 1×10 12 Spraying was performed with a spore suspension of 0.05% (conidia / mL), while the control group was treated with the same amount of 0.05% ( Tween-80 sterile water. Select the consistent growth of lemon pot seedlings for the experiment, spray different concentrations of spore suspension on the leaves of lemon, both sides are sprayed to have liquid drops flow down, after the lemon leaves slightly dry with a net cover (60 cm x 60 cm x 100 cm) cover the lemon seedlings, then 30 head of diuraphis citri adult insects are introduced into the net cover, then spray the spore suspension again. Each concentration of lemon seedlings one pot, three times repeated 5 concentrations, a total of 15 pots. After spraying spore suspension 1, 3, 5, 7 d, respectively, the number of live insects in each treatment is investigated, and the number of live insects on the front and back of the leaves is carefully checked, and the control effect of each treatment is calculated.

[0048] 2 Experimental results The field control effect of different concentrations of Alternaria alternata JY-4 spore suspension on diuraphis citri adults is shown in Figure 4 . After spraying Alternaria alternata JY-4 spore suspension for 1 d, the field corrected mortality of diuraphis citri adults treated with different concentrations of spore suspension was low; after spraying Alternaria alternata JY-4 spore suspension for 3 d, 1 x 10 1 , 1 x 10 3 , 1 x 10 6 , 1 x 10 9 , 1 x 10 12 conidia / mL concentration of spore suspension on diuraphis citri adults was 2.27%, 4.45%, 5.68%, 6.82%, 17.05%; after spraying Alternaria alternata JY-4 spore suspension for 5 d, 1 x 10 9 , 1 x 10 12 conidia / mL concentration of spore suspension on diuraphis citri adults was 18.82%, 23.53%; after spraying Alternaria alternata JY-4 spore suspension for 7 d, 1 x 10 9 , 1 x 10 12 conidia / mL concentration of spore suspension on diuraphis citri adults was 39.76%, 45.78%, respectively, which was 20.94%, 22.25% higher than that after spraying for 5 d.

[0049] As shown in Table 3, after spraying spore suspension for 7 d under field conditions, the LC 50 of diuraphis citri adults treated with different concentrations of Alternaria alternata JY-4 spore suspension was 11.64 x 10 11 conidia / mL; when the concentration was 1 x 10 12 conidia / mL, the LT 50 of diuraphis citri adults treated with Alternaria alternata JY-4 spore suspension under field conditions was 7.52 d (Table 4).

[0050] Table 3 Regression equation of pathogenicity of Alternaria alternata JY-4 to adult of Dialeuroides citri after 7 days of infection

[0051] Table 4 1×10 12 conidia / mL of Alternaria alternata JY-4 to adult of Dialeuroides citri

[0052] In summary, the present application finds that the LC 50 and LT 50 of Alternaria alternata JY-4 to adult of Dialeuroides citri show biological control potential for prevention and control of Dialeuroides citri. 12 After 7 days of treatment with 1×10 50 conidia / mL of Alternaria alternata JY-4 spore suspension, the mortality rate of adult of Dialeuroides citri is 50.00%; the LC 50 and LT 7 of Alternaria alternata JY-4 after 7 days of treatment are 7.89×10 9 conidia / mL and 6.11 days. The test shows that Alternaria alternata JY-4 has certain insecticidal activity to adult of Dialeuroides citri. The results of the present application show that Alternaria alternata JY-4 has a control effect on adult of Dialeuroides citri.

[0053] The present application verifies the control effect of the biocontrol bacteria on Dialeuroides citri through field net test, and the results show that when the spore concentration is 1×10 9 , 1×10 12 conidia / mL, the field corrected mortality rate of Alternaria alternata JY-4 spore suspension to adult of Dialeuroides citri after 7 days of infection of adult of Dialeuroides citri is 39.76% and 45.78% respectively. Under semi-field conditions, the LT 50 of Alternaria alternata JY-4 spore suspension of 1×10 12 conidia / mL sprayed on adult of Dialeuroides citri is 7.52 days, which is significantly slower than the laboratory determination result, which is presumably related to the difficult-to-control environmental factors such as temperature, humidity and light in the field. The next step can be to explore the culture conditions of Alternaria alternata JY-4 and the pathogenic mechanism of infection of Dialeuroides citri.

[0054] The entomogenous fungi separated from the bodies of aphid mummies is identified as Alternaria alternata JY-4 by morphological and molecular biological identification. Figure 5 Alternaria iridiaustralis According to the comprehensive judgment of indoor virulence determination and field control effect test, Alternaria alternata JY-4 has a control effect on adult of Dialeuroides citri.

[0055] Obviously, the above embodiments of the present application are merely exemplary but not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, it is not necessary or possible to enumerate all the embodiments. Any modification, equivalent replacement and improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A strain of Alternaria iridiaustralis JY-4, characterized in that, The strain was preserved in China General Microbiological Culture Collection Center on August 11, 2025, the address of the preservation center is No. 1, Xibaheyan Road, Yard No. 3, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 42162.

2. Microbial inoculant characterized in that, comprising the compound of claim 1 Alternaria iridiaustralis JY-4.

3. The method of claim 1 Alternaria iridiaustralis Use of JY-4 or the microbial inoculant of claim 2 in the preparation of a plant pest control agent for controlling plant pests caused by H. aurantii.

4. Use according to claim 3, characterized in that, The plant includes citrus.

5. A method of controlling plant infestation by Diuraphis noxia by applying to the plant, plant part, or the locus thereof a composition comprising a compound of Formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof. The use according to claim 1 Alternaria iridiaustralis The plant is treated by spraying with the microbial inoculant of JY-4 or claim 2.

6. The method of claim 5, wherein, The plant includes citrus.

7. A product for controlling plant infestation by Diuraphis noxia, characterized in that, comprising the compound of claim 1 Alternaria iridiaustralis JY-4 or the microbial inoculant of claim 2.

8. The product of claim 7, wherein, Alternaria iridiaustralis Alternaria iridiaustralis Spore concentration of JY-4 was 1 x 10 1 ~ 1 x 10 12 conidia / mL.

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