New trichoderma T40 and application thereof in prevention and treatment of plum postharvest brown rot

Abstract

The invention relates to the technical field of microorganisms, in particular to a new trichoderma T40 strain and application thereof in preventing and treating brown rot of harvested plums. The preservation number of the Trichoderma sp. T40 is CCTCC (China Center for Type Culture Collection) NO: M 20251918, and the Trichoderma sp. T40 can be used for preparing the Trichoderma sp. Experimental data show that the trichoderma T40 not only can inhibit the growth of pathogenic bacteria, but also can relieve the harm of brown rot to plum fruits. Compared with traditional chemical bactericides, the trichoderma T40 has obvious environmental protection advantages, does not cause negative effects on the environment after being used, and does not cause harm to human health. Due to the characteristic, the trichoderma T40 has remarkable advantages in the aspects of ecological friendliness and food safety, and is an ideal biological control resource especially in modern green agriculture and organic food production.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a new species of Trichoderma T40 and its application in the prevention and control of postharvest brown rot in plums. Background Technology

[0002] Plum( Prunus salicina As a typical stone fruit, plums are rich in nutrients and have a unique flavor, making them popular with consumers worldwide. However, plums are susceptible to various diseases during post-harvest storage and transportation, significantly affecting their quality and market value. This is especially true for plums caused by diseases such as those affecting the fruit itself. Monilinia fructicola Postharvest brown rot caused by pathogens has become a major hidden danger to the plum industry, posing a serious threat to the marketability and storage life of the fruit.

[0003] Currently, traditional methods for controlling postharvest brown rot in plums mainly rely on chemical pesticides. However, with the emergence of drug-resistant pathogens and increasing concern about the potential environmental and food safety impacts of chemical agents, the sustainability of chemical control has been greatly challenged. Biological control, with its advantages of safety, environmental friendliness, low toxicity, and low resistance, is gradually becoming an ideal alternative for controlling postharvest diseases in fruits and vegetables. By applying harmless probiotics or natural microorganisms, biological control can effectively inhibit the growth of pathogens, reduce the use of chemical pesticides, protect the ecological environment, and is harmless to human health.

[0004] In the field of biological control research, Trichoderma harzianum ( Trichoderma harzianum Bacillus subtilis ( Bacillus subtilis ), fluorescent pseudomonas ( Pseudomonas fluorescens Biocontrol strains such as *Trichoderma* have made positive progress in controlling various postharvest diseases of fruits and vegetables. In particular, *Trichoderma* strains have demonstrated excellent control effects in multiple studies due to their various antibacterial mechanisms (such as competitive exclusion, antibiotic secretion, and degradation of pathogen cell walls), and have effectively extended the shelf life of fruits. However, the types and strains of biocontrol bacteria used for the control of postharvest diseases in plums have not yet been fully developed and utilized, and highly efficient biocontrol bacteria resources remain very limited.

[0005] Therefore, developing new, efficient, and stable biocontrol strains, especially those with specific control effects against postharvest brown rot in plums, has become an essential research focus. In-depth exploration of new biocontrol resources can not only provide the plum industry with more efficient disease control methods but also promote the development of green agriculture and contribute to food safety and environmental protection. Summary of the Invention

[0006] In view of the above-mentioned shortcomings in the prior art, the purpose of this invention is to provide a new species of Trichoderma T40 and its application in the prevention and control of postharvest brown rot in plums.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: Firstly, it provides Trichoderma ( Trichoderma sp.)T40, its accession number is CCTCC NO: M 20251918.

[0008] Secondly, it provides microbial inoculants, including Trichoderma ( Trichoderma sp.)T40. Thirdly, it provides Trichoderma ( Trichoderma sp.) T40 or including Trichoderma ( Trichoderma Application of microbial agent (sp.) T40 in the prevention and control of postharvest brown rot in plums.

[0009] Fourthly, provide Trichoderma ( Trichoderma sp.) T40 or including Trichoderma ( Trichoderma Application of microbial inoculant (spe.)T40 in extending the shelf life of plums.

[0010] The beneficial effects of this invention are as follows: 1. Improved Control Efficacy: Trichoderma T40 effectively inhibits the growth of brown rot pathogens. Experimental data shows that in indoor confrontation experiments, the inhibition rate of Trichoderma T40 can reach 86%. This inhibitory effect is significantly better than other common biocontrol strains, indicating that Trichoderma T40 has stronger disease resistance. Furthermore, in plum fruit inoculation experiments, puncture inoculation with Trichoderma T40 inhibited the growth of brown rot lesions, reducing the lesion diameter by 76%; uninoculated inoculation with Trichoderma T40 effectively reduced the severity index of brown rot in plums, reducing disease occurrence by more than 50%, indicating that Trichoderma T40 not only inhibits the growth of pathogens but also reduces the damage of the disease to the fruit. 2. Reduced environmental pollution and ensured safety: Compared with traditional chemical fungicides, Trichoderma T40 has significant environmental advantages. Its use will not have a negative impact on the environment or harm human health. This characteristic gives Trichoderma T40 a significant advantage in terms of eco-friendliness and food safety, making it an ideal biological control resource, especially in modern green agriculture and organic food production.

[0011] 3. Extended Shelf Life: Trichoderma T40 not only effectively controls postharvest brown rot in plums but also extends their shelf life. Experimental data shows that plums treated with Trichoderma T40 are significantly firmer at room temperature than those in the control group, and have lower levels of total soluble solids (TSS), soluble sugars (SS), and titratable acid (TA), indicating that the ripening process is delayed. This characteristic effectively reduces postharvest rot and quality decline, extending the shelf life of plums during distribution and sales.

[0012] 4. Antibacterial effect of antimicrobial substances: Analysis of the sterile filtrate of Trichoderma T40 revealed an inhibition rate of nearly 50% against brown rot fungus. Trichoderma T40 secretes a variety of substances with antimicrobial activity, which effectively inhibit the growth and reproduction of brown rot fungus. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the plate confrontation analysis between Trichoderma T40 and brown rot fungus; Figure 2 Images of Trichoderma T40 colonies, hyphae, sporulation structures, and spore morphology; Figure 3 A schematic diagram of a phylogenetic tree constructed based on the rpb2 and tef1 genes; Figure 4 A schematic diagram showing the effect of Trichoderma T40 sterile filtrate on the growth of brown rot pathogens; Figure 5 Schematic diagram of inoculating Trichoderma T40 on puncture wounds to control postharvest brown rot in plums; Figure 6 Schematic diagram of the method for controlling postharvest brown rot of plum by inoculating Trichoderma T40 without injury; Figure 7 The plum firmness statistics are shown for the blank control group (CK), the T40 treatment group (T40), the T40 treatment group inoculated with brown rot fungus (T40+Mf), and the brown rot fungus group (Mf). Figure 8 The TSS (Total Saturation Level) of plums in the blank control group (CK), the T40 treatment group (T40), the T40 treatment group inoculated with brown rot fungus (T40+Mf), and the brown rot fungus group (Mf) are statistically analyzed. Figure 9 SS statistics of plums in blank control group (CK), T40 treatment group (T40), T40 treatment group inoculated with brown rot fungus (T40+Mf) and brown rot fungus group (Mf); Figure 10 TA statistical graphs of plums in the blank control group (CK), T40 treatment group (T40), T40 treatment group inoculated with brown rot fungus (T40+Mf), and brown rot fungus group (Mf); Figure 11 Statistical graph of VC in plums for blank control group (CK), T40 treatment group (T40), T40 treatment group inoculated with brown rot fungus (T40+Mf) and brown rot fungus group (Mf). Detailed Implementation

[0014] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0015] Example 1: Screening of Trichoderma strains: Soil samples were taken from a 3m diameter area around a healthy plum tree. 10g of the mixed soil sample was placed in an Erlenmeyer flask containing 90 mL of sterile PBS buffer and incubated in a constant-temperature shaker at 28℃ and 150 r·min. -1 Shake for 30 min under sterile conditions, then dilute with sterile PBS to a concentration of 10. -5 The soil sample was diluted 100 times, and then 100 μL of the prepared diluted solution was spread evenly on a PDA plate and then incubated in a constant temperature incubator at 28℃. The resulting fungal colonies were purified multiple times.

[0016] Select purified fungi and M. fructicola The strains were subjected to confrontation culture on sterile PDA plates, and the strain with the highest antibacterial rate, *Trichoderma*, was screened out. Trichoderma sp.)T40 ( Figure 1 It has an antibacterial rate of up to 86%.

[0017] Example 2: Trichoderma ( Trichoderma Morphological identification of strain T40 (sp.): Use a sterile scalpel to pick up the bacterial colony tissue and place it on a glass slide with a drop of sterile water. Cover with a glass slide and press it down. Observe the morphological characteristics of the hyphae, spores, and other tissue structures, including color, shape, and size, using an optical microscope, and take photos to record the observations. Figure 2 The conidiophores are erect, with a main axis 120-180 μm long and 3.5-4.5 μm wide at the base, forming typical conical branches at the apex. Lateral branches are arranged symmetrically in whorls, each whorl having 3-5 secondary branches. Conidia size: 2.9–3.6 × 2.6–3.4 μm. These morphological characteristics are similar to those of the genus *Trichoderma* (…). Trichoderma The typical characteristics are highly consistent.

[0018] Trichoderma ( Trichoderma Molecular biological identification of strain T40 (sp.): After extracting DNA from the strain, the reaction system consisted of 10 μL PCR buffer mix, 7 μL ddH2O, 1 μL template DNA, and 1 μL each of forward and reverse primers. ITS, rbp2, and tef1 gene fragments were amplified under different reaction conditions (Table 1). After sequencing the products, an ML phylogenetic tree was constructed by combining the rpb2 and tef1 sequences. Figure 3 The strain formed separate branches (ML / PP = 94 / 1), and morphological comparison revealed that the conidia were relatively large; therefore, it was identified as a new species of the genus *Trichoderma*. Trichoderma sp.).

[0019] Strain preservation name: Trichoderma ( Trichoderma sp.) T40; accession number: CCTCC NO: M 20251918; depositary institution: China Center for Type Culture Collection; deposit date: September 1, 2025.

[0020] Table 1 Example 3: Sterile filtrate antibacterial experiment: A suspension of Trichoderma T40 spores cultured at 28℃ and 180 rpm for 7 days on PDB was centrifuged at 12000 rpm for 10 min. The supernatant was filtered through a microbial filter for sterilization. PDA plates were prepared at a ratio of culture medium to sterile filtrate of 2:1. Plates with a diameter of 6 mm were then placed on the plates. M. fructicola The mycelial cake was placed in the center of an agar plate and incubated at 28°C. The colony diameter was recorded using calipers, and the inhibition rate was close to 50%. Figure 4 ).

[0021] Example 4: Trichoderma T40 spore suspension: Using freshly cultured T40 strain (7 days old), add 10 mL of sterile water, gently scrape off colonies with a spreader, filter the mycelia through sterile gauze to collect the spore suspension, and then adjust the concentration of the spore suspension to approximately 1 × 10⁻⁶ with sterile water. 10 Spores / mL. Select plum fruits of uniform size, without mechanical damage, and with relatively consistent maturity. Surface disinfect with 0.5% sodium hypochlorite (NaClO) for 5 min, rinse three times with sterile water, and air dry. Using a sterile punch, make a circular wound with a diameter of 5 mm and a depth of 5 mm at the equator of each plum fruit. Then, inoculate the wound with 20 μL of Trichoderma T40 spore suspension. One day later, inoculate the wound with 5 mm diameter spores. M. fructicola Fungal cakes. Another group had 20 μL of LB medium added to the wound as a control, and were incubated at 28℃ for 2 days. Each treatment had 6 biological replicates, and the experiment was repeated 3 times. The diameter of the lesions was measured daily using a crossover method. Trichoderma T40 inhibited the growth of brown rot lesions, reducing the lesion diameter by 76%. Figure 5 ).

[0022] Example 5: Prepare the same Trichoderma T40 spore suspension as in the experiment above, and immerse the surface-sterilized plum fruits in approximately 1 × 10⁻⁶ mol / L solution. 10 The control group was immersed in a Trichoderma T40 spore suspension at spore density (spores / mL) for 15 min, while the control group was immersed in LB medium for 15 min; after air-drying for 1 h, they were then immersed in... M. fructicola (1 × 10) 10 Spores were incubated in a spore suspension (spores / mL) for 15 min at 28℃ for 3 days. Each treatment had 6 biological replicates, and the experiment was repeated 3 times. The control efficacy of *Trichoderma* T40 against postharvest plum brown rot was evaluated using disease severity. Five levels were used: Level 1 represented 0% diseased area; Level 2 represented 1-25% diseased area; Level 3 represented 26-50% diseased area; Level 4 represented 51-75% diseased area; and Level 5 represented 76-100% diseased area. The Disease Severity Index (DSI) was calculated using the following formula: DSI = ∑(n1×1 + n2×2 + n3×3 + n4×4 + n5×5)×100 / N×5. Where n1, n2, n3, n4, and n5 represent the sample size for each level, and N is the total sample size. The biocontrol effect of Trichoderma T40 inoculated without damage was evaluated using the disease severity index. The results showed that Trichoderma T40 could effectively reduce the severity index of brown rot in plums and reduce the occurrence of the disease by more than 50%. Figure 6 ).

[0023] Example 6: Plum fruits were randomly divided into four treatment groups: (1) control group (CK), in which the fruits were soaked in LB medium for 15 min; (2) Trichoderma T40 (T40) group, in which the fruits were soaked in approximately 1 × 10⁻⁶ LB medium. 10 (3) 15 min in a Trichoderma T40 spore suspension at spore / mL; M. fructicola (Mf) group: Fruits were soaked in LB medium for 15 min, air-dried at room temperature for 1 h, and then placed in... M. fructicola Spore suspension (approximately 1 × 10⁻⁶) 10 (4) Trichoderma T40+M. fructicola Group (T40+Mf), the fruit was soaked in Trichoderma T40 (1 × 10⁻⁶). 10 Spores ( / mL) were immersed in a spore suspension for 15 min, air-dried for 1 h, and then soaked in... M. fructicola (1 × 10) 10Spores were incubated in a spore suspension (spores / mL) for 15 min at 28°C for 3 days. Each treatment was repeated 6 times. Fruit firmness was measured at the equatorial region of the fruit using a GY-4 fruit firmness tester. Figure 7 The soluble solids (TSS) content was determined using a digital refractometer. Figure 8 Hardness and TSS measurements were performed in five biological replicates. Soluble sugar (SS) content was determined using a kit. Figure 9 Titrable acidity (TA) is determined using acid-base titration. Figure 10 Vitamin C (VC) content was quantitatively analyzed using the molybdenum blue colorimetric method. Figure 11 All the above fruit quality indicators were measured in three biological replicates. The results showed that plums treated with Trichoderma T40 had significantly higher firmness at room temperature than those in the control group, and lower contents of soluble solids (TSS), soluble sugars (SS), and titratable acid (TA).

[0024] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0025] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Trichoderma ( Trichoderma sp.)T40, characterized in that, Its accession number is CCTCC NO: M 20251918.

2. A microbial inoculant, characterized in that, Including the Trichoderma as described in claim 1 ( Trichoderma sp.)T40.

3. The Trichoderma as described in claim 1 ( Trichoderma Application of the microbial agent as described in sp.) T40 or claim 2 in the prevention and control of postharvest brown rot in plums.

4. The Trichoderma as described in claim 1 ( Trichoderma Application of the microbial agent as described in sp.) T40 or claim 2 in extending the shelf life of plums.

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