Trichoderma rhododendron GUDJ16 and application thereof

Abstract

The invention discloses trichoderma rhododendron GUDJ16 and application thereof, and belongs to the technical field of microorganisms. The preservation number of the screened trichoderma rhododendron GUDJ16 is CCTCC (China Center For Type Culture Collection) NO: M 20252004. The trichoderma rhododendron GUDJ16 has a prevention and treatment effect on the soft rot of the kiwi fruit, the bacteriostasis rate of the trichoderma rhododendron GUDJ16 on botryosphaeria dothidea can reach 77.15%, and the trichoderma rhododendron GUDJ16 also has a bacteriostasis effect on fusarium solani, fusarium graminearum, fusarium asiaticum, rhizoctonia solani, sclerotium rolfsii, colletotrichum gloeosporioides, phaseolus vulgaris and alternaria tenuissima; the verification of the disease prevention effect on the in-vitro fruits infected with the kiwi fruit soft rot shows that the prevention and treatment effect on the kiwi fruit soft rot after the trichoderma rhododendron GUDJ16 is used for treatment can reach 61.26%, so that the trichoderma rhododendron GUDJ16 has high application value in the field of prevention and treatment of the kiwi fruit soft rot.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a Trichoderma rhododendron GUDJ16 and application thereof. BACKGROUND

[0002] The soft rot of kiwifruit is caused by pathogenic fungi, which seriously threatens the fruit safety during the mature period and postharvest storage of kiwifruit. Studies have shown that Botryosphaeria dothidea is one of the main pathogenic fungi. The soft rot of kiwifruit on the diseased fruit shows that the fruit skin collapses, the tissue softens and is accompanied by rotten odor. After peeling the epidermis of the lesion, a white area is exposed in the center, surrounded by a yellow-green transition zone, and the fruit flesh tissue shows softening and rotting symptoms. Vertical sectioning can see that the soft rot area extends to the inside of the fruit flesh in a conical shape, which is easy to spread and spread.

[0003] Trichoderma spp. are widely present in soil, plant rhizosphere and other natural environments, and have good antagonistic effect on many plant pathogenic fungi, and are an important representative in the research of biocontrol fungi. SUMMARY

[0004] The purpose of the present application is to provide a Trichoderma rhododendron GUDJ16 and application thereof to solve the problems existing in the prior art. The present application isolates and screens a Trichoderma rhododendron GUDJ16, which has the advantages of good prevention and control effect on the soft rot of kiwifruit and high application value in the field of soft rot prevention of kiwifruit.

[0005] To achieve the above purpose, the present application provides the following solutions.

[0006] In a first aspect, the present application provides a Trichoderma rhododendron GUDJ16, which was preserved in the China Center for Type Culture Collection on September 11, 2025, the preservation address is Wuhan University in Wuhan, China, and the preservation number is CCTCC NO: M 20252004.

[0007] In a second aspect, the present application further provides a spore suspension containing spores of the Trichoderma rhododendron GUDJ16.

[0008] In a third aspect, the present application further provides a preparation method of the spore suspension, which comprises the steps of inoculating the Trichoderma rhododendron GUDJ16 into a culture medium and culturing for 5 days, then eluting the culture to obtain the spore suspension.

[0009] In a fourth aspect, the present application further provides a use of the Trichoderma guchi GUDJ16 or the spore suspension in the preparation of a microbial inoculant.

[0010] In a fifth aspect, the present application further provides a microbial inoculant comprising the Trichoderma guchi GUDJ16 or the spore suspension.

[0011] In a sixth aspect, the present application further provides a use of the Trichoderma guchi GUDJ16, the spore suspension or the microbial inoculant in the prevention and treatment of plant pathogenic fungi.

[0012] Preferably, the plant pathogenic fungi are selected from one or more of Botryosphaeria dothidea, Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Aphanomyces euteiches, Colletotrichum musae, Passalora fulva and Alternaria tenuissima.

[0013] In a seventh aspect, the present application further provides a use of the Trichoderma guchi GUDJ16, the spore suspension or the microbial inoculant in the preparation of a product for preventing and treating plant pathogenic fungi.

[0014] In an eighth aspect, the present application further provides a product for preventing and treating plant pathogenic fungi, comprising the Trichoderma guchi GUDJ16, the spore suspension or the microbial inoculant.

[0015] The plant pathogenic fungi are selected from one or more of Botryosphaeria dothidea, Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Aphanomyces euteiches, Colletotrichum musae, Passalora fulva and Alternaria tenuissima.

[0016] In a ninth aspect, the present application further provides a method for preventing and treating plant pathogenic fungi, comprising the step of applying the Trichoderma guchi GUDJ16, the spore suspension or the microbial inoculant to plants.

[0017] The plant pathogenic fungi are selected from one or more of Botryosphaeria dothidea, Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Aphanomyces euteiches, Colletotrichum musae, Passalora fulva and Alternaria tenuissima.

[0018] The present application has the following technical effects:

[0019] The application screens and separates a Trichoderma rhododendron GUDJ16, which has the advantages that the Trichoderma rhododendron GUDJ16 has good prevention and treatment effects on soft rot of kiwifruit, the bacteriostatic rate of the Trichoderma rhododendron GUDJ16 on Botryosphaeria dothidea can reach 77.15 %, and the Trichoderma rhododendron GUDJ16 also has bacteriostatic effects on Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Sclerotium rolfsii, Colletotrichum musae, Passalora fulva and Alternaria tenuissima; it is found through verification of the disease prevention and treatment effects on the in vitro fruit infected with the soft rot of kiwifruit that the prevention and treatment effect of the Trichoderma rhododendron GUDJ16 on the soft rot of kiwifruit can reach 61.26 % after treatment, and the Trichoderma rhododendron GUDJ16 has high application value in the field of prevention and treatment of the soft rot of kiwifruit.

[0020] Collection information: The Trichoderma rhododendron GUDJ16 is preserved in the China Center for Type Culture Collection on September 11, 2025, the preservation address is Wuhan University in Wuhan, China, and the preservation number is CCTCC NO: M20252004. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0022] Figure 1 The figure is the bacteriostatic effect of the Trichoderma rhododendron GUDJ16 screened by the application on Botryosphaeria dothidea; wherein, A is a single B. dothidea growth plate, and B is a colony and B. dothidea co-culture growth plate;

[0023] Figure 2 The figure is the morphological characteristics of the Trichoderma rhododendron GUDJ16 screened by the application; wherein, A is a colony growth on PDA medium, B is a colony growth on SNA medium, C is a colony growth on CMA medium, D is a colony conidium, and E-I is a colony conidium and phialide; the scale of the figure D-I is 10 μm;

[0024] Figure 3 The figure is a phylogenetic tree constructed by a maximum likelihood method through multiple genes (rpb2 and tef1) sequences;

[0025] Figure 4The Trichoderma arizonicum GUDJ16 screened by the application has a prevention and treatment effect on in-vitro fruits infected with soft rot of kiwifruit; wherein, A-C are negative control groups, D-F are positive control groups, and G-I are Trichoderma arizonicum GUDJ16 treatment groups;

[0026] Figure 5 The Trichoderma arizonicum GUDJ16 volatile substance has an inhibitory effect on B. dothidea; wherein, A-B are control groups, and C-D are treatment groups;

[0027] Figure 6 The Trichoderma arizonicum GUDJ16 has an antibacterial effect and an antibacterial rate on eight kinds of pathogenic bacteria. DETAILED DESCRIPTION

[0028] The detailed description of the various exemplary embodiments of the application is not to be considered as limiting the application, but rather as a description of certain aspects, features, and embodiments of the application.

[0029] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range of values, and any other stated value or stated range of values within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0030] 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 application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0031] Various modifications and changes can be made to the specific implementation of the present application described in this specification without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0032] As used herein, the terms "comprise", "comprising", "including", "include", "contain", "containing", "have", "having" or variants thereof are open-ended, and include the stated integer or group, but also any other integer or group.

[0033] Example 1

[0034] 1. Experimental materials

[0035] 1.1 Soil sample

[0036] Rhizosphere soil of Rhododendron anthopogon was collected from Wumeng grassland (104°37'E, 26°11'N) in Liupanshui City, Guizhou Province.

[0037] 1.2 Test target pathogenic bacteria

[0038] Botryosphaeria dothidea was provided by Tobacco College of Guizhou University in Guiyang City, Guizhou Province.

[0039] 1.3 Test culture medium

[0040] PDA culture medium: peeled potato 200 g, glucose 20 g, agar powder 15-20 g, sterile distilled water 1 L, natural pH, sterilized at 121°C for 20 min for standby.

[0041] Low-nutrient agar medium (SNA): KH2PO4 1.0 g, KNO3 1.0 g, MgSO4·7H2O 0.5 g, KCl 0.5 g, glucose 0.2 g, sucrose 0.2 g, agar 12.0 g, distilled water to 1.0 L, sterilized at 121°C for 20 min for standby.

[0042] Corn meal agar medium (CMA): corn steep powder 7.0 g, agar 15.0 g, pH 6.0 ± 0.2, distilled water to 1.0 L, sterilized at 121°C for 20 min for standby.

[0043] 2. Experimental method

[0044] 2.1 Isolation, purification and preservation of strains

[0045] Soil dilution plate coating method (this method is a routine method, which will not be described here) was used to isolate and purify 41 strains from the collected soil, which were placed in 15-20% glycerol cryotubes and stored in a 4°C refrigerator.

[0046] 2.2 Screening of biocontrol strains

[0047] Botryosphaeria dothidea was used as an indicator strain, and the plate confrontation method was used for screening of biocontrol strains. The isolated and screened strains and the bacterial cake of the indicator pathogenic bacteria were inoculated in PDA culture medium 2 cm apart, and only the bacterial cake of the indicator was inoculated as a control, with 3 replicates for each treatment. It was placed in a 28°C constant temperature incubator in the dark and observed regularly for the growth of the strains, and the growth was recorded by taking pictures.

[0048] The bacteriostatic rate calculation formula is: bacteriostatic rate = [(C-T) / C] x 100%.

[0049] Wherein C and T represent the average growth radius of the pathogenic bacteria in the control group and the treatment group, respectively.

[0050] 2.3 Identification of biocontrol strains

[0051] 2.3.1 Morphological observation

[0052] The strains obtained by screening were inoculated in PDA medium and cultured in a constant temperature incubator at 28°C in the dark. The growth performance of the strains on the medium was observed during the culture, including the colony color and sporulation condition. Then the morphological characteristics such as conidiophore, conidium (n=50) and sporulation structure were observed under a Carl Zeiss Microscopy GmbH (Jena, Germany) microscope at 40 times magnification (1x40).

[0053] 2.3.2 Molecular biological identification

[0054] The genomic DNA of the screened biocontrol strains was extracted using Ezup column fungal genomic DNA extraction kit from Shanghai Biomed. The ITS universal primer pair (ITS4 and ITS5), RPB2 primer pair (fRPB2-5f and fRPB2-7cr) and TEF1 primer pair (EF1-728F and TEF1LLErev) synthesized by Beijing Qikong Biotechnology (Chongqing) Co., Ltd. The specific primer information is shown in Table 1.

[0055] Table 1 Primer information

[0056]

[0057] Polymerase chain reaction (PCR) was performed in a 25 μL reaction volume containing 1 μL DNA template, 1 μL forward primer, 1 μL reverse primer, 12.5 μL 2x Tap Plus MaserMix, and 9.5 μL sterile water.

[0058] The PCR products were purified and sequenced by Beijing Qikong Biotechnology Co., Ltd. (Chongqing).

[0059] The RPB2 and TEF1-a sequences of 32 other T. strains and 2 outgroup species, Protocrea farinosa CBS 121551 and P. pallida CBS 299.78, were downloaded from GenBank (see Table 2) and used to construct a phylogenetic tree using the maximum likelihood (ML) method. ML analysis was performed on the IQ-TREE Web server (http: / / iqtree.cibiv.univie.ac.at) with 1000 rapid bootstrap replicates under partitioned models, and the phylogenetic tree was visualized by FigTree v.1.4.0.

[0060] Table 2. Strain information

[0061]

[0062] Note: T and ET in the strain number represent that the strain is a type strain.

[0063] 3. Biocontrol efficacy of biocontrol bacteria on detached kiwifruit fruit

[0064] Preparation of biocontrol bacteria spore suspension: the screened biocontrol bacterial strains were inoculated on PDA medium plates and cultured in a constant temperature incubator at 25°C for 5 days. Then, the culture was eluted with sterile water to prepare a spore suspension with a concentration of 1 x 10 6 spores / mL for standby.

[0065] The "Guichang" kiwifruit variety was selected. Healthy fruits with uniform shape, size, and maturity were selected. A stab wound was made at the equatorial region of the fruit, and 50 μL of a biocontrol bacterial spore suspension with a concentration of 10 6 spores / mL was added to the wound and allowed to dry. A fresh P. expansum bacterial cake with a diameter of 3 mm was taken using a puncher and placed on the kiwifruit wound. The positive control was inoculated with only the pathogenic bacterial cake, and the negative control was inoculated with a sterile PDA cake with sterile water. The treated fruits were placed in a constant temperature incubator at 25°C, and the fruit disease was observed, and the lesion diameter was measured. The experiment was repeated three times, and after 7 days of inoculation, the size of the leaf lesion was measured using a vernier caliper. The disease control effect on detached kiwifruit fruit was calculated according to the following formula: Disease control effect (%) = [(diameter of lesion in control group - diameter of lesion in treatment group) / diameter of lesion in control group] x 100%.

[0066] 4. Inhibition of biocontrol bacterial volatile products

[0067] The inhibitory activity of volatile metabolites of the screened biocontrol strains on Botryosphaeria dothidea was evaluated by the flat plate confrontation method. The biocontrol strain and pathogen were inoculated in the center of a PDA plate with a diameter of 5 mm, respectively. The plate inoculated with the pathogen was inverted on the plate inoculated with the biocontrol strain, and the gap between the two plates was sealed. The control group was only inoculated with the pathogen and cultured in the same way. All treatments were placed in a constant temperature incubator at 25°C. When the control pathogen grew over the plate, the cross method was used to measure the colony diameter of the pathogen in the treatment group, and the inhibition rate was calculated as [(C-5)- (T-5) / (C-5)] x 100%. Where C and T represent the diameters of the measured pathogen in the control and treatment groups, respectively.

[0068] 5. Inhibition spectrum of biocontrol bacteria

[0069] Eight different fungal pathogens (Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Sclerotium rolfsii, Colletotrichum musae, Diaporthephaseolorum, and Alternaria tenuissima) were selected for the determination of the inhibition spectrum of biocontrol strains using the flat plate confrontation method. The inhibition rate was calculated according to "3. Method for determining the control effect of biocontrol bacteria on kiwifruit soft rot in vitro".

[0070] The above eight fungal pathogens were preserved by the Plant Pathology Teaching and Research Office of Guizhou University in Guiyang, Guizhou Province.

[0071] 6. Results analysis

[0072] 6.1 Isolation and screening of biocontrol strains for kiwifruit soft rot

[0073] The inhibition effect of Trichoderma strains on B. dothidea was determined by the flat plate confrontation method using B. dothidea as the indicator. The results are shown in Figure 1 and Table 3. One Trichoderma strain with good inhibition effect on B. dothidea was finally screened and named GUDJ16, with an inhibition rate of 77.15%.

[0074] Table 3 Inhibition effect of biocontrol strains on Botryosphaeria dothidea on pathogen

[0075] Treatment Colony size (mm) Inhibition rate CK 58.46 ± 0.29 b ]] - Treatment 13.36 ± 0.95 a ]] 77.15±1.62

[0076] Note: Different letters in the table represent significant difference (P<0.05), as below.

[0077] 6.2 Morphological identification

[0078] The strain GUDJ16 with good antibacterial effect on Botryosphaeria dothidea isolated by the previous screening was identified by morphology.

[0079] The strain grew full plate on PDA, SNA and CMA medium after 5 days, and the colony morphology on different media was found to have great difference.

[0080] On PDA medium, the colony was slightly cottony, and the mycelium was tight. Green conidia formed thick and dense concentric rings. The spores near the cake were more dense and darker in color. The colony did not produce pigment, and had a faint coconut flavor; on SNA medium, the colony was willow-like, with sparse aerial mycelium, and conidia formed thin and dispersed concentric rings. The spore color was yellow-green, and the color near the cake was green; on CMA medium, the aerial mycelium was dense, and the conidia were thick and dense, covering the entire plate.

[0081] The strain could produce conidiophores and conidia on PDA medium at 28℃ after 7 days of culture. The conidiophores had obvious main shafts, with paired or unilateral lateral branches arranged closely; the phialides were long-necked bottle-shaped, mostly 2-3 whorled, occasionally solitary, with a size of 5.09-11.10 μm×2.3-4.31 μm. The conidia were single-celled, spherical, with a size of 3.1-3.9 μm×3.15-3.78 μm, and appeared yellow-green under the microscope, without thick-walled chlamydospores.

[0082] 6.3 Multigene phylogenetic analysis

[0083] The sequences of 35 strains including the strain screened by the application were subjected to multi-site phylogenetic analysis. The analysis used rpb2 and tef1 gene tandem sequences, and Protocrea farinosa CBS 121551 and P. pallida CBS 299.78 as outgroups. In the maximum likelihood (ML) system tree, all strains formed a monophyletic group with a high statistical support value, and the strain GUDJ16 formed an independent branch. Comparison found that the colony morphology of T. simile was quite different from that of GUDJ 16, and no chlamydospore was found in GUDJ 16, and the colony morphology of T. guizhouense was quite different from that of GUDJ16, and the molecular spore of GUDJ16 was larger. Combined with morphological and molecular biological identification, it was identified as a new species of Trichoderma, Trichoderma rhododendron.

[0084] Trichoderma rhododendron GUDJ16 was preserved in China Center for Type Culture Collection on September 11, 2025, and the address of preservation is Wuhan University in Wuhan, China, and the preservation number is CCTCC NO: M 20252004.

[0085] 6.4 The prevention and treatment effect of Trichoderma rhododendron GUDJ16 on detached kiwifruit fruit of soft rot

[0086] The results are shown in Figure 4 and Table 4. After 7 days of treatment, no disease symptoms were observed in the negative control group, and disease symptoms were observed in the positive control group and the treatment group. The average lesion diameter of the positive control group was 43.49 mm, and the average lesion diameter of the treatment group was 16.85 mm. There was a significant difference between the two groups. The prevention and treatment effect of Trichoderma rhododendron GUDJ16 screened by the application on kiwifruit soft rot was 61.26%. Therefore, the Trichoderma rhododendron GUDJ16 screened by the application has excellent prevention and treatment effect on kiwifruit soft rot.

[0087] Table 4 Prevention and treatment effect of different treatment groups on detached kiwifruit fruit of soft rot

[0088] Treatment Lesion diameter (mm) Relative control efficiency (%) Negative control group - - Positive control group 43.49 ± 1.77 b ]] - GUDJ16 16.85 ± 0.09 a ]] 61.26±0.21

[0089] 6.5 Inhibition effect of volatile substances of Trichoderma rhododendron GUDJ16 on B. dothidea

[0090] The results show that Figure 5 and Table 5, the volatile substances of Trichoderma rhododendron GUDJ16 have a significant inhibition effect on the mycelial diameter of B. dothidea, and also have an inhibition effect on the spore production of B. dothidea on PDA plate.

[0091] Table 5. Inhibition rate of volatile substances from Trichoderma rhododendron GUDJ16 on hyphal diameter of B. dothidea

[0092] Colony diameter (mm) Bacteriostatic rate (%) CK 83.65 ± 0.35 b ]] - GUDJ16 52.75 ± 0.41 a ]] 39.23±0.52

[0093] 6.6 Broad-spectrum antibacterial activity of Trichoderma rhododendron GUDJ16

[0094] See results Figure 6 Through experiments on the inhibition of mycelial growth of eight pathogenic fungi, the results showed that Trichoderma rhododendron GUDJ16 had an inhibition rate of over 53% against all pathogens. Among them, the inhibition rate against Fusarium solani, Fusarium graminearum and Rhizoctonia solani was less than 60%, while the inhibition rate against the other five pathogens was over 65%.

[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of Trichoderma rhododendron (GUDJ16), characterized in that, The aforementioned Trichoderma rhodopsin GUDJ16 was deposited at the China Center for Type Culture Collection (CCTCC) on September 11, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20252004.

2. A spore suspension, characterized in that, The spore suspension contains spores of Trichoderma rhododendron GUDJ16 as described in claim 1.

3. A method for preparing the spore suspension according to claim 2, characterized in that, The procedure includes inoculating the *Trichoderma rhododendron* GUDJ16 into a culture medium and culturing it for 5 days, then eluting the culture to obtain the spore suspension.

4. The use of the Trichoderma rhododendron GUDJ16 as described in claim 1 or the spore suspension as described in claim 2 in the preparation of microbial inoculants.

5. A microbial inoculant, characterized in that, The microbial agent includes Trichoderma rhododendron GUDJ16 as described in claim 1 or the spore suspension as described in claim 2.

6. The application of Trichoderma rhododendron GUDJ16 as described in claim 1, the spore suspension as described in claim 2, or the microbial agent as described in claim 5 in the control of plant pathogens.

7. The application according to claim 6, characterized in that, The plant pathogens are selected from one or more of the following: Staphylococcus aureus, Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Sclerotium sclerotiorum, Banana anthracnose fungus, Bean intercropping scabies, and Alternaria spp.

8. The use of Trichoderma rhododendron GUDJ16 as described in claim 1, the spore suspension as described in claim 2, or the microbial agent as described in claim 5 in the preparation of products for controlling plant pathogens.

9. A product for preventing and controlling plant pathogens, characterized in that, The product includes Trichoderma rhododendron GUDJ16 as described in claim 1, the spore suspension as described in claim 2, or the microbial agent as described in claim 5; The plant pathogens are selected from one or more of the following: Staphylococcus aureus, Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Sclerotium sclerotiorum, Banana anthracnose fungus, Bean intercropping scabies, and Alternaria spp.

10. A method for controlling plant pathogens, characterized in that, The method includes the step of applying the Trichoderma rhododendron GUDJ16 of claim 1, the spore suspension of claim 2, or the microbial agent of claim 5 to the plant; The plant pathogens are selected from one or more of the following: Staphylococcus aureus, Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Sclerotium sclerotiorum, Banana anthracnose fungus, Bean intercropping scabies, and Alternaria spp.

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