Chaetomium formosum and application of chaetomium formosum in prevention and treatment of pine wood nematode disease

Abstract

The invention discloses chaetomium formosanum and application thereof in prevention and treatment of pine wood nematode disease, and belongs to the technical field of biocontrol of plant diseases. The name of the bacterial strain is Chaetomium ascotrichioides 1-24-2, and the preservation number of the bacterial strain is GDMCC No: 64397. The invention further discloses a preparation method of the chaetomium ascotrichioides strain. The chaetomium formosanum 1-24-2 disclosed by the invention is derived from resistant masson pine plant tissues; the chaetomium formosanum 1-24-2 and the biological agent obtained based on the chaetomium formosanum 1-24-2 have a strong inhibition effect on pine wood nematode diseases, are low in requirements on culture conditions and have good development and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease biocontrol, and discloses a Chaetomima scotrichoides 1-24-2 and application thereof in preventing and controlling pine wood nematode disease. Background Art

[0002] Pine wilt disease (PWD), also known as pine wilt, is a major quarantine disease of pine trees caused by the pine wood nematode (Bursaphelenchus xylophilus) (Yang Baojun et al., 2003; Zhu Yunqian et al., 2021; Yang Li et al., 2024; Dong Baoxu, 2023; Hong Chenghao et al., 2023; Zhang Yamin, 2021; National Forestry and Grassland Administration, 2023). Pine wilt can infect 106 host species, 60 of which are naturally susceptible (He Longxi et al., 2011). Pinus massoniana is an important afforestation species in my country's subtropical regions, characterized by rapid growth, high yields, excellent wood properties, high resin production, and strong adaptability. The host plants and vector beetles of the pine wilt nematode are widely distributed in my country, covering almost all regions of the country, providing a biological basis for the spread of pine wilt disease. At present, physical and chemical control methods are mainly adopted for pine wood nematodes. However, these methods are only suitable for small areas and are not very practical for large areas of pine forests. Chemical pesticides are highly toxic and highly residual, which can pollute the air, water and soil environment and indirectly cause harm to humans. In addition, because trees injected with pesticides will rot after being felled, they lose their economic value.

[0003] To this end, numerous studies have been devoted to developing safe and effective biological agents. Screening microorganisms with nematicidal activity from resistant Masson pine plant tissues is of great practical significance, as it aims to provide a theoretical basis for the development of plant tissue microbial resources and the utilization of endophytes that kill pine wood nematodes.

[0004] Chaetomium is a widely distributed fungus found in soil, on dead leaves and wood, as well as in healthy plants as an endophyte, causing diseases in some plants (Zhang Weining, 2017). Chaetomium fungi produce a rich array of secondary metabolites, including ergosterol, chrysanthellol, and chaetoglobosins. These metabolites possess potent cytotoxic, antibacterial, and antifungal activities (Hu Yang, 2013). Numerous studies have demonstrated that treatment with Chaetomium culture filtrates alone exhibits potent nematocidal activity against nematodes. Yan Xiaoning (2010) isolated a Chaetomium strain with a 100% lethality against southern root-knot nematodes within 12 hours. Zhang Kaiyuan (2017) reported that 11 Chaetomium strains exhibited varying degrees of antagonism against four maize pathogens: corn leaf spot, crescent spore, corn round spot, and corn leaf spot. Zhang Yun et al. (2020) reported that a Chaetomium globosum strain can effectively control cotton Verticillium wilt. Its mechanism of action primarily involves antibiotic activity and induced resistance, suggesting promising application prospects in cotton Verticillium wilt control. Zhang Leiming (2016) identified two Chaetomium fungi with significant antagonistic effects against the pathogenic fungus of ginseng black spot. Therefore, Chaetomium fungi, as an important natural resource, can be used for the development of biocontrol strains (Tian & Li, 2022). Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a Chaetomium ascotrichoides 1-24-2 strain having pine wood nematode killing activity.

[0006] Another object of the present invention is to provide a biological preparation prepared from the above-mentioned Chaetomium caenorhabditis 1-24-2, which has high lethal activity against pine wood nematodes.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned biological preparation.

[0008] Another object of the present invention is to provide applications of the above-mentioned Chaetomium caenorhabditis 1-24-2 and its metabolites.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A Chaetomium ascotrichoides strain, named Chaetomium ascotrichoides 1-24-2, was isolated and purified from healthy Masson pine tissue.

[0011] The preservation information of the Chaetomium ascotrichoides 1-24-2 is as follows: the preservation unit is Guangdong Microbial Culture Collection Center (GDMCC), the preservation time is March 7, 2024, the preservation address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, the preservation number is GDMCC No: 64397.

[0012] The Chaetomium 1-24-2 fungus forms irregular, round colonies on PDA culture medium. The main body is yellow-green with white edges, and there are fewer aerial hyphae with yellow droplets attached to the hyphae. Ascospores are lemon-shaped or nearly spherical, with a protrusion at each end. Mature ascospores are olive-colored, measuring approximately 6.0-9.0 μm by 5.1-6.8 μm, with an average size of 7.0 ± 0.7 μm by 6.0 ± 0.4 μm.

[0013] A biological preparation comprises at least one of the fermentation liquid, fermentation filtrate, fermentation supernatant and spore suspension of the above-mentioned Chaetomium caenorhabditis 1-24-2.

[0014] Furthermore, the fermentation broth is prepared by liquid fermentation of the Chaetomium caenorhabditis 1-24-2, preferably comprising the following steps: inoculating Chaetomium caenorhabditis 1-24-2 into PDB culture medium for cultivation to obtain the fermentation broth.

[0015] Preferably, the pH value of the PDB culture medium is 6.0-10.0.

[0016] Preferably, the culture refers to shaking culture at 25°C to 30°C with a speed of 120 to 200 r / min for 3 to 7 days; more preferably, the culture refers to shaking culture at 25°C to 28°C with a speed of 160 to 180 r / min for 3 to 7 days.

[0017] Furthermore, the fermentation filtrate is prepared by filtering the fermentation broth;

[0018] Preferably, the filtration is performed using a microporous filter membrane with a pore size of 0.22 μm.

[0019] Furthermore, the fermentation supernatant is prepared by centrifuging the fermentation broth.

[0020] Preferably, the centrifugation condition is 8000-10000 rpm for 15-20 min; more preferably, 10000 rpm for 15 min.

[0021] The present invention also provides the application of the above-mentioned Chaetomium caenorhabditis 1-24-2 or biological preparation, which is one or more combinations of the following applications:

[0022] (1) Application in the prevention and control of pine wood nematode disease.

[0023] (2) Application in the preparation of preparations for preventing and treating pine wood nematode disease;

[0024] (3) Application in killing pine wood nematodes;

[0025] (4) Application in the preparation of preparations for killing pine wood nematodes.

[0026] The present invention has the following advantages and effects compared to the prior art:

[0027] (1) The Chaetomium caenorhabditis 1-24-2 obtained by the present invention is derived from resistant Masson pine plant tissues and has long-term mutualistic symbiosis with Masson pine, thus avoiding the possibility of destroying the stability of the microecological environment on the plant surface;

[0028] (2) The Chaetomium 1-24-2 of the present invention and the biological preparation obtained based on it have a strong inhibitory effect on pine wood nematode disease and have good development and application prospects.

[0029] (3) The Chaetomium caenorhabditis 1-24-2 obtained by the present invention has low requirements for culture conditions and has good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the front view of the colony of strain 1-24-2 on PDA medium;

[0031] Figure 2 This is the front view of the colony of strain 1-24-2 on PDA medium;

[0032] Figure 3 This is the hyphae morphology of strain 1-24-2;

[0033] Figure 4 This is the spore morphology of strain 1-24-2;

[0034] Figure 5 The effect of different liquid volumes of strain 1-24-2 on the activity of pine wood nematode;

[0035] Figure 6 The effect of different fermentation times of strain 1-24-2 on the activity of pine wood nematodes;

[0036] Figure 7 The effect of different fermentation temperatures of strain 1-24-2 on the activity of pine wood nematodes;

[0037] Figure 8 This is the effect of different initial fermentation pH values ​​of strain 1-24-2 on the activity of pine wood nematodes. DETAILED DESCRIPTION

[0038] The present invention will be described in further detail below in conjunction with Examples and accompanying drawings, but embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art. Unless otherwise specified, the reagents and materials used in the present invention can be obtained commercially.

[0039] Various culture medium formulas:

[0040] (1) PDA culture medium: Weigh 200 g of peeled potatoes, cut into thin slices, add 1 L of ultrapure water, boil for 20-25 min, filter out solids with 4 layers of gauze, add 20 g of glucose and 15 g of agar powder to the collected liquid, dissolve thoroughly and adjust the volume to 1 L, divide into Erlenmeyer flasks, sterilize at 121°C for 20 min, and dry for later use.

[0041] (2) PDB culture medium: Weigh 200 g of peeled potatoes, cut into thin slices, add 1 L of ultrapure water, and boil for 20-25 min. Filter out the solids with 4 layers of gauze. Add 20 g of glucose to the collected liquid, dissolve it completely, and adjust the volume to 1 L. After dispensing into Erlenmeyer flasks, sterilize at 121°C for 20 min, and dry for later use.

[0042] (3) NA culture medium: Weigh 33.0 g of NA sample, dissolve it in 1000 mL of ultrapure water by heating, dispense it into Erlenmeyer flasks, sterilize it by high pressure at 121°C for 15 min, and dry it for later use.

[0043] Example 1: Isolation and purification of Chaetomium caenorhabditis 1-24-2

[0044] Healthy side branches of Masson pine (Pinus massoniana) approximately 10 cm long and 0.5–0.7 cm in diameter were cut into cylindrical blocks approximately 1 cm long and 0.5–0.7 cm in diameter using sterilized pruning shears. These blocks were treated with 75% alcohol for 30 seconds, rinsed three times with sterile water, soaked in 10% sodium hypochlorite for 3 minutes, and rinsed four more times with sterile water.

[0045] The surface disinfection efficacy was verified using the rinse test: 100 μL of the final sterile water rinse was applied to the PDA culture medium as a control, and repeated three times. After incubating the treated PDA plate at 26 ± 1°C in the dark for 7 days, if no colonies were observed on the plate, the surface disinfection was complete; otherwise, re-isolation was required.

[0046] After surface disinfection, use a sterile utility knife to remove the bark from the Masson pine tissue block. Cut the cylindrical tissue block into several pieces along the cross section. Place the pieces with the longitudinal section facing downward on PDA culture medium using sterile tweezers. Incubate at 25°C for 3–7 days, repeat three times, and record bacterial growth daily. When colonies emerge, pick the top hyphae for purification.

[0047] Example 2: Activity of Chaetomium caenorhabditis 1-24-2 against pine wood nematodes

[0048] The purified strain was inoculated in the center of a PDA plate and cultured at 25°C for 3-5 days. When the mycelium grew to 1 / 2 of the culture dish, 5 mycelium pieces with a diameter of 5 mm were taken along the edge of the colony and transferred to a 500 mL Erlenmeyer flask containing 250 mL of PDB medium. After culturing at 25°C and 160 rpm for 3 days, the fermentation filtrate of the test strain was obtained by filtration.

[0049] The nematocidal activity of isolated fungal culture filtrates was determined using the immersion method. 450 μL of the fermentation filtrate of the test strain and 50 μL of the nematode suspension (containing approximately 50 nematodes) were added to a 48-well plate, gently shaken to mix, and placed in a constant-temperature incubator at 25°C. Nematode survival was observed every 24 hours under an inverted microscope, and the corrected mortality rate was calculated. After 48 hours, an equal volume of sterile water was added to each treatment, the cells were resuspended, and allowed to stand for 5-10 minutes. This was repeated three times to allow nematode recovery. Nematodes were considered dead when they became rigid and showed no response to stimulation with a dissecting needle. An equal amount of uninoculated PDB medium was used as a control, with three replicates for each treatment. After comparative screening, strain 1-24-2 was identified, demonstrating high lethality against pine wood nematodes.

[0050] CM=(dc) / (1-c)×100%;

[0051] Wherein, CM represents the corrected mortality rate, %; c represents the nematode mortality rate in the control group, %; d represents the nematode mortality rate in the treatment group, %;

[0052] Therefore, the strain 1-24-2 isolated in this example has a high lethal activity against pine wood nematodes.

[0053] Example 3: Identification of Chaetomium caenorhabditis 1-24-2

[0054] Strain 1-24-2 was cultured on PDA medium at 25°C. Microscopic examination revealed irregularly round colonies with a yellow-green main body and white edges. There were few aerial hyphae, with yellow droplets attached to the hyphae. Ascospores were lemon-shaped or nearly spherical, with a bulge at each end. Mature ascospores were olive-colored, approximately 6.0-9.0 × 5.1-6.8 μm, with an average size of 7.0 ± 0.7 × 6.0 ± 0.4 μm ( Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 ).

[0055] The strain DNA was extracted and used as a template for PCR amplification using the primers ITS (ITS1\ITS5), tub2 (bt2a\bt2b) and rpb2 (RPB2AM-1bf\RPB2AM-7R) listed in Table 1. The resulting ITS sequence of strain 1-24-2 was 548 bp (SEQ ID No. 1), tub2 sequence was 434 bp (SEQ ID No. 2), and rpb2 sequence was 1019 bp (SEQ ID No. 3).

[0056] Table 1 Primer information

[0057]

[0058] BLAST analysis of the three sequences revealed up to 99% homology to Chaetomium fungi. A multi-gene joint analysis phylogenetic tree constructed with Achaetomium strumarium (CBS 333.67) as an outgroup indicated that the endophytic fungus 1-24-2 was located in the same clade as Chaetomium ascotrichoides, with a bootstrap value (probability) of 99%. Based on the phylogenetic tree results and morphological characteristics of the endophytic fungus 1-24-2, it was identified as Chaetomium ascotrichoides.

[0059] Therefore, the strain 1-24-2 was named Chaetomium ascotrichoides 1-24-2, and the preservation information was as follows: the deposit unit was Guangdong Microbial Culture Collection Center (GDMCC), the preservation time was March 7, 2024, the preservation address was: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and the preservation number was GDMCC No: 64397.

[0060] Example 4: Effects of different culture conditions on the nematicidal activity of fungal strain 1-24-2

[0061] 1. Effects of different liquid volumes on the activity of strain 1-24-2 in killing pine wood nematodes

[0062] PDB culture medium (100 / 250mL, 150 / 250mL, 100 / 500mL, 200 / 500mL, or 300 / 500mL) was prepared in Erlenmeyer flasks. Strain 1-24-2 was inoculated into these flasks and cultured at 28°C, 180 rpm, for 3 days. The fermentation broth was transferred to a 50mL centrifuge tube and centrifuged at 10,000 rpm for 15 minutes. The supernatant was then collected and stored at 4°C for later use. The nematicidal activity of strain 1-24-2 was tested using the immersion assay targeting pine wood nematodes. 450 μL of strain 1-24-2 supernatant and 50 μL of nematode suspension (containing approximately 50 nematodes) were added to a 48-well plate and gently shaken to mix thoroughly. The plate was then placed in a 25°C incubator. Nematode survival was observed every 24 hours using an inverted microscope, and the corrected mortality rate was calculated. An equal amount of uninoculated PDB medium was selected as the control, and 3 replicates were set for each treatment.

[0063] Conclusion: If Figure 5 As shown, the above different liquid volumes can all produce active substances that kill pine wood nematodes, but when the liquid volume is 100 / 250mL, the supernatant of the fermentation liquid of the endophytic fungus 1-24-2 has the best activity in killing pine wood nematodes, with a corrected mortality rate of 96.81% against pine wood nematodes.

[0064] 2. Effects of different fermentation times on the activity of pine wood nematodes

[0065] Inoculate strain 1-24-2 into 100 mL of PDB medium in a 250 mL Erlenmeyer flask. Incubate for 3, 5, 7, 9, 11, and 13 days at 28°C and 180 rpm. Transfer the fermentation broth to a 50 mL centrifuge tube and centrifuge at 10,000 rpm for 15 minutes. Remove the supernatant and store at 4°C until ready for use. Test the nematicidal activity of strain 1-24-2 using the pine wood nematode immersion assay, following the same procedure as in step 1 above.

[0066] Conclusion: If Figure 6 As shown, on the 3rd day of fermentation, the supernatant of the fermentation liquid of endophytic fungus 1-24-2 had the best activity in killing pine wood nematodes, with a corrected mortality rate of 88.78% against pine wood nematodes. After 9th day, the activity of the supernatant of the fermentation liquid of endophytic fungus 1-24-2 in killing pine wood nematodes decreased significantly.

[0067] 3. Effects of different fermentation temperatures on the activity of pine wood nematodes

[0068] Strain 1-24-2 was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of PDB medium. The flask was placed in a shaker at 20°C, 24°C, 28°C, 32°C, and 36°C at 180 rpm for 3 days. The fermentation broth was transferred to a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 15 minutes. The supernatant was collected and stored at 4°C until ready for use. Nematicidal activity of strain 1-24-2 was tested using the immersion assay targeting pine wood nematodes, following the same procedure as in step 1 above.

[0069] Conclusion: If Figure 7 As shown, when the temperature was 28℃, the supernatant of the fermentation broth of endophytic fungus 1-24-2 had the best activity in killing pine wood nematodes, with a corrected mortality rate of 85.99% against pine wood nematodes.

[0070] 4. Effects of different initial pH on the activity of pine wood nematodes

[0071] Prepare PDB medium with initial pH values ​​of 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. Inoculate strain 1-24-2 into 100 mL of PDB medium in a 250 mL Erlenmeyer flask and incubate at 28°C, 180 rpm, and incubate for 3 days. Transfer the fermentation broth to a 50 mL centrifuge tube and centrifuge at 10,000 rpm for 15 minutes. Remove the supernatant and store at 4°C until ready for use. Test the nematicidal activity of strain 1-24-2 using the immersion method targeting pine wood nematodes, following the same procedure as in step 1 above.

[0072] Conclusion: If Figure 8 As shown, when the pH was 6, the supernatant of the fermentation broth of endophytic fungus 1-24-2 had the best activity in killing pine wood nematodes, with a corrected mortality rate of 99.70% against pine wood nematodes.

[0073] References:

[0074] Yang Baojun, Pan Hongyang, Tang Jian, et al. Pine wood nematode disease[M]. China Forestry Publishing House, 2003.

[0075] Yan Xiaoning, A. Sikora R., Zheng Jingwu. Different effects of seven cucumber endophytic fungi on the second instar larvae of the southern root-knot nematode [J]. Chinese Journal of Biological Control, 2010, 26(2): 181-185.

[0076] He Longxi, Wu Xiaoqin, Yu Luzhen. Relationship between differences in superoxide radicals and pathological changes in the interaction between different pine trees and pine wood nematodes[J]. Journal of Nanjing Forestry University (Natural Science Edition), 2011, 35(02): 25-30.

[0077] Zhang Weining. Classification and identification of antagonistic Chaetomium 61239 and its biocontrol effect[D]. Northwest Agriculture and Forestry University, 2017.

[0078] Hu Yang. Molecular biology research on secondary metabolism of Chaetomium globosum[D]. Nankai University, 2013.

[0079] Zhang Yamin. The five-year national campaign for pine wood nematode disease prevention and control has been fully launched[J]. National Land Greening, 2021, (07): 39.

[0080] Zhang Leiming, Xu Jiao, Liu Zhenpeng, et al. Screening of antagonistic bacteria against ginseng black spot[J]. Journal of Northeast Forestry University, 2016, Vol. 44(11): 89-91.

[0081] Zhang Kaiyuan, Tang Baohong. Screening of antagonistic Chaetomium endophytes against corn leaf spot[C] / / Mycological Society of China. Abstracts of the 7th National Congress of the Mycological Society of China and the 2017 Annual Academic Meeting. College of Life Sciences, Zhengzhou University; 2017:1.

[0082] Zhang Yun. Study on the inhibitory mechanism of Chaetomium globosum CEF-082 against cotton Verticillium dahliae[D]. Nanjing Agricultural University, 2020.001963.

[0083] Zhu Yunqian, Huang Weiyan, Xie Weilong, et al. Research progress on prevention and control methods of pine wood nematode disease[J]. Hebei Forestry Science and Technology, 2021(2):44-48.

[0084] National Forestry and Grassland Administration. Announcement No. 7 of 2023 of the National Forestry and Grassland Administration - Announcement on Pine Wood Nematode Infected Areas in 2023 [Z]. 2023.

[0085] Yang Li, Xu Xiaoji. Prevention and control strategies for the risk of introduction of pine wood nematodes[J]. Xinjiang Agricultural Reclamation Science and Technology, 2024, 47(01): 33-36.

[0086] Dong Baoxu. Characteristics of pine wood nematode damage and integrated control techniques[J]. Modern Horticulture, 2023, 46(16): 73-75.

[0087] Hong Chenghao, Zhao Fei, Yu Weiguo, et al. Research progress on stem injection control technology for pine wood nematode disease[J]. Southern Forestry Science, 2023, 51(02): 71-74.

[0088] Tian Y,Li Y(2022)A review on bioactive compounds from marine-derivedChaetomium species.J Microbiol Biotechnol 32(5):541.https: / / doi.org / 10.4014 / jmb.2201.01007.

[0089] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A strain of Chaetomium caecum, characterized by: The name is Chaetomium ascotrichoides 1-24-2, and it was deposited in the Guangdong Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province on March 7, 2024, with the deposit number: GDMCC No: 64397.

2. A biological agent, characterized in that: The biological preparation comprises at least one of the fermentation broth, fermentation filtrate, fermentation supernatant and spore suspension of the Chaetomium caenorhabditis 1-24-2 according to claim 1.

3. The biological agent according to claim 2, characterized in that: The fermentation liquid is prepared by liquid fermentation of the Chaetomium caenorhabditis 1-24-2 described in claim 1.

4. The biological agent according to claim 3, characterized in that: The following steps are involved: The fungus 1-24-2 was inoculated into PDB medium for cultivation to obtain a fermentation broth.

5. The biological agent according to claim 4, characterized in that: The pH value of the PDB culture medium is 6.0-10.0; The culture refers to shaking culture at 25°C to 30°C and 120 to 200 r / min for 3 to 7 days.

6. The biological agent according to any one of claims 2 to 6, characterized in that: The fermentation filtrate is prepared by filtering the fermentation broth.

7. The biological agent according to claim 6, characterized in that: The filtration is performed using a microporous filter membrane with a pore size of 0.22 μm.

8. The biological agent according to any one of claims 2 to 6, characterized in that: The fermentation supernatant is prepared by centrifuging the fermentation broth.

9. The biological agent according to claim 8, characterized in that: The centrifugal conditions are 8000-10000 rpm for 15-20 min.

10. Use of the Chaetomium caerulea according to claim 1 or the biological agent according to any one of claims 2 to 9, characterized in that: One or more combinations of the following applications: (1) Application in the prevention and treatment of pine wood nematode disease; (2) Application in the preparation of preparations for preventing and treating pine wood nematode disease; (3) Application in killing pine wood nematodes; (4) Application in the preparation of preparations for killing pine wood nematodes.

Images