A strain of Trichoderma subviride and its application in controlling plant diseases

Abstract

The present invention discloses a strain of Trichoderma paraviridescens and its application in controlling plant diseases, belonging to the technical field of plant disease control. The preservation number of the Trichoderma paraviridescens is CGMCC No. 41126. The present invention also discloses the application of the Trichoderma paraviridescens or a broad-spectrum antibacterial agent containing the Trichoderma paraviridescens in inhibiting plant pathogenic bacteria. The Trichoderma paraviridescens provided by the present invention has a significant antagonistic effect against Diplodia pinea, the pathogen causing dead twig disease of Masson pine, and other common plant pathogenic bacteria, is environmentally friendly and pollution-free, provides a safe and reliable new way for the control of various plant diseases, and has important significance in the field of biological control of plant diseases. Moreover, the Trichoderma paraviridescens provided by the present invention is simple to culture, easy to preserve, has a wide antibacterial spectrum, is convenient for industrial production, and has good development and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of plant disease control, and particularly to a strain of Trichoderma subviride and its application in controlling plant diseases. Background Art

[0002] As an important native tree species in China, Pinus massoniana Lamb. has a wide distribution range, covering central and southern China. It is an important pioneer tree species for afforesting barren mountains and a constructive tree species in the forest ecosystem south of the Yangtze River. It plays an important role in national greening and ecological security construction. It is also an important timber tree species in southern China, with high economic value and wide uses. Pine shoot blight is the main disease that harms Pinus massoniana. The pine shoot blight pathogen was first discovered in 1823 and named Sphaeria sapinea. After several inspections and revisions, the pathogen was corrected to Diplodia sapinea (Fr.) Fuckel, belonging to the Ascomycota, Dothideomycetes, Botryosphaeriales, Botryosphaeriaceae, and Diplodia. The pine shoot blight pathogen generally has a latent infection phenomenon on Pinus massoniana, causing the main shoots of the host trees to wither, the branches to ulcerate, and the growth to be hindered. In severe cases, it can even kill the whole tree. Pine shoot blight has caused serious harm and significant losses to pine plantations.

[0003] At present, the prevention and control of pine tip blight mainly rely on chemical control, forest cultivation, and biological control. However, chemical control is extremely likely to cause environmental pollution, and forest cultivation is a relatively long process. With the development of biotechnology and the increasing concern about environmental safety among people, using biological control methods to prevent and control pine tip blight is a relatively effective prevention and control measure. With the rise of the concept of biological control, using microbial strain resources to prevent and control plant diseases has become a new trend in the development of disease prevention and control. Trichoderma species are typical biocontrol agents, which inhibit the development of pathogenic bacteria by producing antagonistic substances, competing for nutrients and ecological niches, and hyperparasitism. At present, the role of Trichoderma strains in preventing and controlling soil-borne diseases is the most prominent, and they can effectively prevent and control root rot, damping-off, and Phytophthora blight. They also have a strong prevention and control effect on medicinal plant diseases and have strong antagonistic effects against Fusarium equiseti, the root rot pathogen of Astragalus membranaceus, Sclerotinia sp., the sclerotinia pathogen of Glehnia littoralis, and Phytophthora cactorum, the damping-off pathogen of Panax quinquefolius. However, there are no relevant reports on the antagonism of Trichoderma paraviridescens against multiple pathogenic bacteria such as Diplodia pinea, Ophiostoma bicolor, Endoconidiophora fujiensis, E. polonica, Gnomoniopsis daii, the chestnut fruit rot pathogen, Gnomoniopsis daii, and Gnomoniopsis chinensis, the chestnut canker pathogen. Summary of the Invention

[0004] The purpose of the present invention is to provide a strain of Trichoderma paraviridescens and its application in preventing and controlling plant diseases to solve the problems existing in the above-mentioned prior art. This Trichoderma paraviridescens has a significant antagonistic effect against Diplodia pinea and other common plant pathogenic bacteria, and has a wide antibacterial spectrum. It can be used as a biocontrol preparation for various plant diseases, providing a new way for the biological control of plant diseases.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides a strain of Trichoderma paraviridescens, which is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The preservation time is March 21, 2024, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is CGMCC No. 41126.

[0007] The present invention also provides a broad-spectrum antibacterial agent, which contains the strain or sterile fermentation broth of the above-mentioned Trichoderma paraviridescens.

[0008] Furthermore, the broad-spectrum antibacterial agent includes an antibacterial agent against Diplodia sapinea (Fr.) Fuckel, Ophiostoma bicolor, Endoconidiophora fujiensis, Endoconidiophora polonica, Gnomoniopsis daii, or Gnomoniopsis chinensis.

[0009] The present invention also provides the use of the aforementioned Trichoderma subviride in the preparation of a broad-spectrum antibacterial agent, which includes an antibacterial agent against Diplodia sapinea (Fr.) Fuckel, Ophiostoma bicolor, Endoconidiophora fujiensis, Endoconidiophora polonica, Gnomoniopsis daii, or Gnomoniopsis chinensis.

[0010] The present invention also provides the use of the aforementioned Trichoderma subviride or the broad-spectrum antibacterial agent in inhibiting plant pathogenic bacteria, which includes Diplodia sapinea (Fr.) Fuckel, Ophiostoma bicolor, Endoconidiophora fujiensis, Endoconidiophora polonica, Gnomoniopsis daii, or Gnomoniopsis chinensis.

[0011] The present invention also provides a method for controlling plant diseases, which includes the step of applying the aforementioned Trichoderma subviride or the broad-spectrum antibacterial agent to plants to inhibit the growth of disease-causing bacteria.

[0012] Furthermore, the plant diseases include plant diseases caused by Diplodia sapinea (Fr.) Fuckel, Ophiostoma bicolor, Endoconidiophora fujiensis, Endoconidiophora polonica, Gnomoniopsis daii, or Gnomoniopsis chinensis.

[0013] Furthermore, the plant diseases caused by Gnomoniopsis daii include chestnut fruit rot or oak leaf spot.

[0014] The present invention discloses the following technical effects:

[0015] The Trichoderma subviride provided by the present invention has a significant antagonistic effect against Diplodia pinea, the pathogen causing pine shoot blight, is environmentally friendly and pollution-free, and is of great significance for the prevention and control of pine shoot blight. This strain has a significant antagonistic effect against common plant pathogens (Ophiostoma bicolor, Endoconidiophora fujiensis, Endoconidiophora polonica, Gnomoniopsis daii or Gnomoniopsis chinensis), is environmentally friendly and pollution-free, inhibits the growth of pathogenic fungi, provides a safe and reliable new approach for the prevention and control of various plant diseases, and is of great significance in the field of biological control of plant diseases.

[0016] The Trichoderma subviride provided by the present invention is easy to culture, easy to preserve, has a wide antibacterial spectrum, is convenient for industrial production, and has good development and application prospects. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 Morphological characteristics of the Trichoderma subviride strain; A: Front of the strain; B: Back of the strain; C: Conidiophores; D: Conidia;

[0019] Figure 2 Phylogenetic tree of the Trichoderma subviride strain based on tef1 (A) and rpb2 (B) gene sequences;

[0020] Figure 3 Results of the plate confrontation experiment between Trichoderma subviride and Diplodia pinea; A: Control group inoculated only with Diplodia pinea; B: Treatment group inoculated with Diplodia pinea and Trichoderma subviride, with Diplodia pinea on the left and Trichoderma subviride on the right;

[0021] Figure 4 Inhibitory effect of the sterile filtrate of Trichoderma subviride on the pathogen of pine shoot blight; A: Control group of Diplodia pinea; B: Treatment group of co-culturing the sterile filtrate of Trichoderma subviride and Diplodia pinea;

[0022] Figure 5 Figure showing the inhibitory effect of the sterile filtrate of Trichoderma subviride on other pathogenic fungi; A: Ophiostoma bicolor (CK); B: Co-culture of the sterile filtrate of Trichoderma subviride and Ophiostoma bicolor; C: Endoconidiophora fujiensis (CK); D: Co-culture of the sterile filtrate of Trichoderma subviride and Endoconidiophora fujiensis; E: Endoconidiophora polonica (CK); F: Co-culture of the sterile fermentation broth of Trichoderma subviride and Endoconidiophora polonica; G: Gnomoniopsis daii, the causative agent of chestnut fruit rot (CK); H: Co-culture of the sterile filtrate of Trichoderma subviride and Gnomoniopsis daii, the causative agent of chestnut fruit rot; I: Gnomoniopsis daii, the causative agent of oak leaf spot (CK); J: Co-culture of the sterile filtrate of Trichoderma subviride and Gnomoniopsis daii, the causative agent of oak leaf spot; K: Gnomoniopsis chinensis, the causative agent of chestnut canker (CK); L: Co-culture of the sterile filtrate of Trichoderma subviride and Gnomoniopsis chinensis, the causative agent of chestnut canker.

[0023] Figure 6 Figure showing the safety test effect of inoculating the antagonistic fungus Trichoderma subviride into three-year-old Masson pine; A: Positive control group inoculated with Diplodia pinea; B: Treatment group inoculated with Trichoderma subviride.

[0024] Figure 7 Figure showing the results of the incidence of Masson pine shoot blight controlled by Trichoderma subviride; A: Only inoculated with Diplodia pinea (14 days); B: Only inoculated with Diplodia pinea (21 days); C: Co-inoculated with Diplodia pinea + Trichoderma subviride (14 days); D: Co-inoculated with Diplodia pinea + Trichoderma subviride (21 days). Detailed implementation manners

[0025] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0029] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0030] Example 1 Isolation and Identification of Trichoderma subviride

[0031] In the early stage of the present invention, a conventional fungal isolation method was used to isolate strain CXY4102 from the branches and trunks of Masson pine suffering from shoot blight in Chongqing in August 2019.

[0032] Morphological characteristic identification of strain CXY4102: The strain was cultured on PDA medium for 4 days, and the colony diameter was about 9 cm ( Figure 1 ). The mycelia of this strain grew radially, without producing aerial mycelia. The color of the mycelia was white in the initial stage and gradually changed to light yellow with the extension of time. The conidia were green. The conidiophores were short with many pairs of small branches, with round or oval conidia, scattered or dense, clustered at the top of the conidiophores, and the size of the conidia was (2.56 - 4.29) × (1.64 - 2.52) μm.

[0033] The tef1 and rpb2 DNA sequences of strain CXY4102 were obtained by sequencing. By blasting the measured tef1 and rpb2 DNA sequences, closely related strain sequences with very high homology could be found in GenBank. The sequence with the highest similarity to strain CXY4102 was the Trichoderma paraviridescens sequence, with a homology of over 99% in both cases. The ML tree was constructed using the software RAxML-HPC v.7.0.3, with the general time reversible (GTR) GAMMA model selected, and the bootstrap support rate was calculated by repeating 1000 iterative calculations. The software FigTree v.1.4.3 and Adobe Illustrator CS6 were used to edit and beautify the constructed phylogenetic tree. The results showed that strain CXY4102 (represented by the deposit number CGMCC 41126 in the figure) and Trichoderma paraviridescens clustered on the same branch ( Figure 2 ). Combining with the morphological characteristics of the strain on PDA medium, strain CXY4102 was identified as Trichoderma paraviridescens.

[0034] The Trichoderma paraviridescens CXY4102 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on March 21, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 41126.

[0035] Example 2 Verification of the inhibitory effect of Trichoderma paraviridescens on Diplodia pinea, the pathogen of pine shoot blight, by plate confrontation

[0036] Diplodia pinea, the pathogen of pine shoot blight (stored in the Key Laboratory of the National Forestry and Grassland Administration - Forest Pathology Research Laboratory, Chinese Academy of Forestry), was used as the indicator strain. The pathogen and the Trichoderma paraviridescens strain were activated 5 days in advance for use. 6-mm-diameter pathogen and antagonist discs were punched from the edge of the vigorously growing colonies on 2% MEA medium and inoculated on the left and right sides of a disposable plastic Petri dish respectively. The two discs were 5 cm apart through the center of the Petri dish. The Petri dish inoculated only with Diplodia pinea was used as the control, and there were 5 replicates for each treatment. The inoculated Petri dishes were inverted and cultured in an incubator at 25 °C to observe whether the strain had an antagonistic effect on the pathogen. The growth radius of the pathogen colony pointing to the antagonist was measured every day to calculate the inhibition rate. Inhibition rate (%) = (control colony radius - treatment colony radius) / control colony radius × 100.

[0037] Confrontation culture of Trichoderma paraviridescens and Diplodia pinea (Figure 3 ) Among them, the inhibition of Trichoderma subviride on Diplodia pinea is proportional to time. The inhibition rate of Trichoderma subviride on Diplodia pinea was 63.33% after 4 days of confrontation culture (Table 1). Trichoderma subviride grows rapidly and quickly occupies the growth space to cover the pathogen.

[0038] Table 1 Inhibitory rate of Trichoderma subviride on Diplodia pinea after 4 days of confrontation culture

[0039]

[0040] Example 3 Inhibitory effect of the sterile fermentation broth of Trichoderma subviride strain on Diplodia pinea

[0041] The Trichoderma subviride strain was activated on PDA medium for 5 days. A 6-mm punch was used to cut out a mycelial disc and placed it in a 250-mL Erlenmeyer flask containing 100 mL of PDB medium. It was cultured on a constant temperature shaker (26 °C, 180 r / min) for 5 days to obtain a fungal fermentation broth. Subsequently, the fermentation broth was centrifuged at 6000 r / min for 5 min, and the supernatant was collected and filtered through a 0.22-μm microporous membrane to remove bacteria, obtaining a sterile filtrate. It was stored in a 4 °C refrigerator for later use. The filtered supernatant was added to the PDA (1:10) medium cooled to 50 - 55 °C, mixed well, and poured into plates. After the plates cooled and solidified, they were used to determine the antibacterial effect of fungal non-volatile substances. Using PDA with the same volume of sterile water added as a control, a 6-mm Diplodia pinea mycelial disc was inoculated in the center of the plate. The growth of the colony was observed regularly every day. When the fungal colony in the control group grew to 3 / 4 of the medium, the colony radius was measured using the cross method, and the inhibition rate was calculated. All treatments were repeated 5 times. The antibacterial effect is shown in Table 2. Figure 4 As shown, the inhibition rate of the sterile fermentation broth of Trichoderma subviride strain on Diplodia pinea was 45.17%. Inhibition rate = (control colony diameter of the pathogen - treated colony diameter) / control colony diameter of the pathogen × 100%.

[0042] Table 2 Inhibitory rate of the sterile fermentation broth of Trichoderma subviride on Diplodia pinea

[0043]

[0044] Example 4 Inhibitory effect of the sterile filtrate of Trichoderma subviride strain on common pathogens

[0045] Referring to Example 3, the mycelial growth rate method was used to measure the antibacterial activities of the sterile filtrate of Trichoderma subviride strain against 6 common fungi such as Biscogniauxia mediterranea (all provided by CFCC, China Forestry Microorganism Preservation Center), and the results are shown in Table 3. Figure 5Overall, the colony diameter of the treatment group was significantly smaller than that of the control group. The strain Trichoderma subviride had the best antibacterial effect against Ophiostoma bicolor, Endoconidiophora fujiensis, and Endoconidiophora polonica, with the highest antibacterial rate reaching 87.02%; it had a general antibacterial effect against the pathogens of chestnut fruit rot and oak leaf spot (Gnomoniopsis daii), with antibacterial rates of 20.29% and 34.01% respectively; it also had a certain antibacterial effect against the chestnut canker pathogen, but the antibacterial effect was not high, with an antibacterial rate of only 7.99%. The study found that the strain Trichoderma subviride not only had an antagonistic effect against Diplodia pinea, the pathogen of pine tip blight, but also had an antibacterial effect against many other important forest pathogenic fungi. However, relatively speaking, it had a better antibacterial effect against the pathogens isolated from coniferous trees, followed by those from broad-leaved trees. The results showed that the strain Trichoderma subviride had a wide antibacterial spectrum and had the potential to control various plant diseases, and could be used as a high-quality biocontrol strain for research.

[0046] Table 3 Antibacterial rates of non-volatile substances of antagonistic fungi against other pathogens

[0047]

[0048] Example 5 Safety evaluation of the strain Trichoderma subviride

[0049] To confirm whether the initially screened strain Trichoderma subviride is safe, the method of inoculation with injury was adopted, and the strain Trichoderma subviride was inoculated onto three-year-old Masson pines with consistent growth. Ten days after inoculation, visually, there were no any disease symptoms on the three-year-old Masson pine seedlings. After peeling off the outer bark of the Masson pine, there were no obvious necrotic spots at the inoculation site of the phloem of the Masson pine inoculated with Trichoderma subviride. However, for the Masson pine seedlings inoculated with the positive control of Diplodia pinea, obvious necrotic spots appeared at the inoculation site, and the size of the necrotic spots was 2.94 ± 1.72 cm 2 ( Figure 6 ) Therefore, this experiment confirmed that the strain Trichoderma subviride was not pathogenic to three-year-old Masson pines.

[0050] Example 6 Control effect of the strain Trichoderma subviride on pine tip blight

[0051] The inoculation method of wounding was used to inoculate *Diplodia pinea* and *Diplodia pinea* + *Trichoderma subviride* on 3-year-old Masson pine seedlings. *Diplodia pinea* and *Trichoderma subviride* were respectively cultured on 2% MEA medium for 7 days under dark conditions at 25 °C for standby. For the control group, 10 Masson pine seedlings with consistent growth vigor were selected. On the tree trunks at 20 cm and 40 cm above the ground, a horizontal hole was drilled into the sapwood of the Masson pine seedlings with a cork borer with a diameter of 6 mm. Subsequently, a 6-mm-diameter mycelial plug from the edge of the vigorously growing *Diplodia pinea* colony on the medium was quickly transferred into the inoculation hole (point) with a disposable sterilized toothpick, and then the bark was covered and fixed with sealing film; similarly, in the experimental group, *Diplodia pinea* and *Trichoderma subviride* were inoculated on the Masson pine seedlings at the same time. Two inoculation points were set for each Masson pine seedling, and each treatment was repeated 5 times. At 14 days and 21 days after inoculation respectively, the bark surface of the Masson pine tree was gently scraped with a utility knife to measure the size of the lesion. And about 0.5 cm 2 samples were taken and re-isolated on 2% MEA medium. By combining the colony morphology of the strains with the DNA sequences, it was verified whether the newly obtained strains were the inoculated pathogens. Control rate = (pathogen lesion area - lesion area of the treatment group) / pathogen lesion area × 100%.

[0052] Twenty-one days after inoculation, the Masson pine did not show obvious withering. After peeling off the outer bark, brown to black necrotic lesions appeared at the inoculation site of *Diplodia pinea*, and the size of the lesion area was 139 ± 7.06 mm 2 , and the necrotic lesion area of the combined inoculation of *Diplodia pinea* and *Trichoderma subviride* was much smaller than that of the single inoculation of *Diplodia pinea* (Table 4, Figure 7 ), and the control rates at 14 days and 21 days after inoculation were 47.06% and 58.99% respectively (Table 4). *Diplodia pinea* was obtained from the diseased part again through tissue isolation at the junction of the diseased and healthy tissues. The combined inoculation treatment separated and cultured *Diplodia pinea* and *Trichoderma subviride* at the same time.

[0053] Table 4 Control effect of *Trichoderma subviride* on Masson pine shoot blight

[0054]

[0055] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Trichoderma paraviridescens, characterized in that: It is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on March 21, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No.41126.

2. A broad-spectrum antibacterial agent, characterized in that: A strain or sterile fermentation liquid comprising the Trichoderma subtilis according to claim 1.

3. The use of Trichoderma subadenoviride in the preparation of a broad-spectrum antibacterial agent as claimed in claim 1, characterized in that: The broad-spectrum antibacterial agent is an antibacterial agent of Diplodia sapinea (Fr.) Fuckel, Ophiostoma bicolor, Endoconidiophora fujiensis, Endoconidiophora polonica, Gnomoniopsis daii or Gnomoniopsis chinensis.

4. Use of the Trichoderma subadenoviride according to claim 1 or the broad-spectrum antibacterial agent according to claim 2 in inhibiting plant pathogens, characterized in that: The plant pathogenic bacteria are Diplodia sapinea (Fr.) Fuckel, Ophiostoma bicolor, Endoconidiophora fujiensis, Endoconidiophora polonica, Gnomoniopsis daii or Gnomoniopsis chinensis.

5. A method for preventing and controlling plant diseases, characterized in that: The method comprises applying the Trichoderma subtilis described in claim 1 or the broad-spectrum antibacterial agent described in claim 2 to plants to inhibit the growth of pathogenic bacteria; The plant disease is a plant disease caused by Diplodia sapinea (Fr.) Fuckel; and the plant is Pinus massoniana.

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