Trichoderma sp.3302 and its application in biocontrol and growth promotion

Abstract

The present application belongs to the field of microbial technology, and discloses a new Trichoderma azadirachtae 3302 strain and application thereof in biocontrol and growth promotion. The Trichoderma azadirachtae 3302 has been preserved in the China General Microbiological Culture Collection Center on May 6, 2024, and the strain preservation number is CGMCC No. 3.27235. The Trichoderma azadirachtae 3302 of the present application is a new species, has the effects of antagonizing Fusarium oxysporum and Sclerotium rolfsii, has a good control effect on related diseases such as banana wilt, and can be used for the control of related diseases of food crops and economic crops; meanwhile, the strain has a good salt stress resistance and a good environmental adaptability; in addition, the strain also has a good effect of promoting plant growth and development, has a significant promoting effect on the germination and root length of rice seeds and the growth of banana seedlings, and has a good application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology. More specifically, it relates to a new species of Trichoderma azadirachtae 3302 and the application of the strain in promoting growth, antagonizing Fusarium oxysporum or Sclerotium rofsii, and preventing related diseases. BACKGROUND

[0002] Trichoderma is an internationally recognized biocontrol microorganism with wide application in the biological control of plant fungal diseases. Moreover, Trichoderma not only can directly inhibit the growth of pathogenic fungi, but also can colonize in plant roots and interact with plants through various mechanisms, thereby directly or indirectly promoting plant growth. Therefore, Trichoderma, as a biocontrol fungus that can inhibit pathogens and promote plant growth, has been widely used.

[0003] Currently, the commercially used Trichoderma for plant disease control mainly includes T. harzianum, T. asperellum and T. atroviride, etc. There is obvious homogeneity in the source of Trichoderma species, which affects the exploration of new functions of biocontrol products and the development of new Trichoderma biopesticides.

[0004] The existing biocontrol Trichoderma has different control effects, and there are problems such as degeneration and variation of microorganisms. Therefore, it is of great significance to develop new Trichoderma strains with biocontrol and growth promotion effects for the development of new biological pesticides, plant protection, biological fertilizer and the prevention of banana wilt and other fields. SUMMARY

[0005] The present application aims to provide a new Trichoderma strain with biocontrol and growth promotion effects. The present application researches and obtains a new species of Trichoderma azadirachtae 3302, which not only has strong inhibitory effect on Fusarium oxysporum and Sclerotium rofsii, but also has good control effect on related diseases, and has salt tolerance, promotes the growth and development of rice and banana, and other functions.

[0006] The above-mentioned object of the present application is achieved by the following technical solutions:

[0007] The present application provides a new Trichoderma azadirachtae 3302, which was preserved in the China General Microbiological Culture Collection Center on May 6, 2024, and the strain preservation number is CGMCC No. 3.27235.

[0008] Further, the tef1-alpha sequence of the Trichoderma azadirachtae 3302 is shown in SEQ ID No. 1,

[0009] Further, the rpb2 sequence of the Trichoderma huillachii 3302 is shown as SEQ ID No. 2.

[0010] The application provides the use of the tef1-α sequence shown as SEQ ID No. 1 or the rpb2 sequence shown as SEQ ID No. 2 in identifying the Trichoderma huillachii 3302. The specific application mode is as follows: the use of a reagent for detecting the tef1-α sequence shown as SEQ ID No. 1 or the rpb2 sequence shown as SEQ ID No. 2 in the preparation of a product for identifying the Trichoderma huillachii 3302.

[0011] The application also includes a culture of the Trichoderma huillachii 3302, which is a substance obtained by culturing the Trichoderma huillachii 3302 in a microbial culture medium.

[0012] Further, the microbial culture medium is a culture medium that can realize the growth and proliferation of the Trichoderma huillachii 3302, including but not limited to PDA, PD culture medium, PDAm Trichoderma selective culture medium, CMD corn flour glucose agar culture medium, SNA low-nutrient agar culture medium, MEA malt juice agar culture medium, and other improved culture media for better growth or metabolism of the Trichoderma huillachii 3302.

[0013] Further, the culture is a spore suspension, a bacterial suspension and / or a fermentation broth of the Trichoderma huillachii 3302.

[0014] Further, the spore suspension is obtained by flushing the spores produced by the Trichoderma huillachii 3302 after being cultured in a PDA solid culture medium, and then filtering the flushing liquid to remove the bacterial bodies.

[0015] Further, the concentration of the spore suspension is 1.0×10 1 to 1.0×10 10 spores / mL, preferably 1.0×10 6 spores / mL.

[0016] Further, the bacterial suspension is obtained by activating the Trichoderma huillachii 3302 in a PDA solid culture medium, then transferring it to a liquid culture medium for culture, filtering the filtrate, then draining and crushing the bacterial bodies, and then adding sterile water.

[0017] Further, the concentration of the bacterial suspension is 1% to 10%, preferably 2%.

[0018] Further, the fermentation broth is obtained by activating the Trichoderma huillachii 3302 in a PDA solid culture medium, then transferring it to a liquid culture medium for culture, filtering the filtrate, and then centrifuging the filtrate to obtain the supernatant, i.e. the fermentation broth of the Trichoderma huillachii 3302. Optionally, the liquid culture medium can be PD culture medium.

[0019] Further, the fermentation liquid concentration is 1%-10%.

[0020] The application further provides a microbial agent or fertilizer containing the Trichoderma azadirachtae 3302 and / or culture of the Trichoderma azadirachtae 3302.

[0021] Further, the microbial agent or fertilizer is in the form of tablets, seed coating agents, dry suspending agents, water dispersible granules or wettable powders.

[0022] The application provides application of the Trichoderma azadirachtae 3302 or the culture of the Trichoderma azadirachtae 3302 or the microbial agent or fertilizer of the Trichoderma azadirachtae 3302 in promoting growth of rice or banana, inhibiting Fusarium oxysporum f.sp.cubense tropical race 4 (FocTR4) or Sclerotium rolfsii Sacc, or preventing plant diseases caused by the Fusarium oxysporum f.sp.cubense tropical race 4 and / or the Sclerotium rolfsii Sacc.

[0023] The application further provides application of the Trichoderma azadirachtae 3302 or the culture of the Trichoderma azadirachtae 3302 or the microbial agent or fertilizer of the Trichoderma azadirachtae 3302 in preparing a product capable of promoting growth of rice or banana, inhibiting Fusarium oxysporum f.sp.cubense tropical race 4 or Sclerotium rolfsii Sacc, and / or preventing plant diseases caused by the Fusarium oxysporum f.sp.cubense tropical race 4 and / or the Sclerotium rolfsii Sacc.

[0024] The application has the following beneficial effects:

[0025] The application separates a new Trichoderma species, i.e., Trichoderma azadirachtae 3302, which has the effects of antagonizing Fusarium oxysporum f.sp.cubense tropical race 4 and Sclerotium rolfsii Sacc, can be used for preventing related diseases of food crops, economic crops and the like, has good salt stress resistance and good environmental adaptability. In addition, the strain also has the effects of promoting growth and development of rice and banana, and has significant promoting effects on seed germination, root length, root system and growth of seedlings, and has good application value. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a morphological feature diagram of the strain 3302. A-D: morphological feature diagrams of the strain 3302 after growing on PDA, MEA, CMD and SNA culture mediums for 7 days; E: spore cluster morphology; F: chlamydospore morphology; G-K: conidial phialide morphology; L-N: conidium morphology.

[0027] Figure 2is a phylogenetic tree of strain 3302 constructed by maximum likelihood method (ML) based on the combined genes of tef1-α, ITS and rpb2.

[0028] Figure 3 is the antagonistic effect of T. arjanai 3302 on FocTR4 and Sclerotium rolfsii. a: CK (FocTR4); b: treatment group (PDA plate left inoculated with T. arjanai 3302, right inoculated with FocTR4) c: CK (Sclerotium rolfsii); d: treatment group (PDA plate left inoculated with T. arjanai 3302, right inoculated with Sclerotium rolfsii).

[0029] Figure 4 is the growth of T. arjanai 3302 under different concentrations of NaCl. A: the growth of T. arjanai 3302 under different concentrations of NaCl for 48h and 120h; B: colony diameter measurement analysis of T. arjanai 3302 under different concentrations of NaCl for 48h and 120h.

[0030] Figure 5 is the growth-promoting effect of T. arjanai 3302 on rice seeds. A: the growth of rice seeds treated with T. arjanai 3302 for 3d; B: germination rate measurement analysis of rice seeds treated with T. arjanai 3302 for 3d; C: root length measurement analysis of rice seeds treated with T. arjanai 3302 for 3d; D: shoot length measurement analysis of rice seeds treated with T. arjanai 3302 for 3d.

[0031] Figure 6 is the growth-promoting effect of T. arjanai 3302 on banana. A: the growth of banana seedlings treated with T. arjanai 3302 for 40d; B: fresh crown weight measurement analysis of banana seedlings; C: fresh root weight measurement analysis of banana seedlings; D: stem diameter measurement analysis of banana seedlings; E: leaf number measurement analysis of banana seedlings; F: plant height measurement analysis of banana seedlings; G: root length measurement analysis of banana seedlings; H: root number measurement analysis of banana seedlings.

[0032] Figure 7 is the external control effect of T. arjanai 3302 on banana Fusarium wilt.

[0033] Figure 8 is the external control effect data analysis of T. arjanai 3302 on banana Fusarium wilt.

[0034] Figure 9 is the internal control effect of T. arjanai 3302 on banana Fusarium wilt.

[0035] Figure 10 is the internal control effect data analysis of T. arjanai 3302 on banana Fusarium wilt. DETAILED DESCRIPTION

[0036] The present application is further described in conjunction with the accompanying drawings and specific examples, which do not limit the present application in any manner. Unless otherwise specified, the reagents, methods, and apparatus used in the following examples are those conventional in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] Example 1 Isolation and identification of Trichoderma azadirachdii 3302

[0039] Trichoderma azadirachdii 3302 was isolated from rhizosphere soil samples of Azadirachta indica A. Juss. plants in Zaoqing, Guangdong Province, and was deposited in the China General Microbiological Culture Collection Center on May 6, 2024, with the accession number CGMCC No. 3.27235.

[0040] The specific isolation and identification process is as follows:

[0041] 1. Isolation of strain 3302

[0042] Rhizosphere soil samples of Azadirachta indica A. Juss. plants of the Meliaceae family in Zaoqing, Guangdong Province were collected. The soil sample collection method used the five-point sampling method, and 100 g of soil at a depth of 10-15 cm below the ground was collected and placed in a sampling sealed bag for storage at 4°C.

[0043] Trichoderma strain isolation: dilution coating method was used, 10 g of dry soil was taken into a 250 mL conical flask, 90 mL of sterile water was added, and it was placed in a shaking bed for 15 min of 28°C and 180 rpm shaking to mix, then taken out, 10 -1 times soil dilution suspension was obtained; 1 mL was taken into a 10 mL centrifuge tube with a pipette, and sterile water was added to make up to 10 mL, that is, 10 -2 times soil dilution suspension; 10 -3 times soil dilution suspension was obtained according to the law. On the clean bench, the above three kinds of soil dilution suspensions (10 -1 , 10 -2 , 10 -3) each 100 μL to PDA Trichoderma selective medium (formula: 200 g potato boiled for 20 min, then filtered, leaving the filtrate, adding 18 g glucose, 18 g agar, 0.08 g chloramphenicol, 0.25 g streptomycin and 0.02 g rose Bengal, and then adding distilled water to make the volume reach 1 L), evenly coated with a spreader, sealed and placed at 28℃, 12 h light and 12 h dark conditions. After three days, the mycelium of the suspected Trichoderma strain was picked on a PDA plate for single mycelium purification, and then a plate confrontation method was used to screen strains with good antibacterial effect on Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4), and finally a strain with good antibacterial effect on FocTR4 was obtained, named 3302.

[0044] 2. Morphological characteristics of strain 3302

[0045] (1) Colony morphology observation:

[0046] Test medium: PDA potato dextrose agar medium (formula: 200 g peeled potato pieces boiled for 30 min, then filtered, leaving the filtrate, adding 18 g glucose, 18 g agar, and then adding distilled water to make up to 1 L);

[0047] CMD corn meal dextrose agar medium (formula: 30 g corn meal boiled for 30 min, then filtered, leaving the filtrate, adding 18 g glucose, 18 g agar, and then adding distilled water to make the volume reach 1 L);

[0048] SNA low-nutrient agar medium (formula: KH2PO4 1 g, KNO3 1 g, MgSO4·7H2O 0.5 g, KCl 0.5 g, glucose 0.2 g, sucrose 0.2 g, agar powder 20 g, and then adding distilled water to make up to 1 L);

[0049] MEA malt extract agar medium (formula: 20 g malt extract, 18 g agar, and then adding distilled water to make up to 1 L).

[0050] A 6 mm diameter puncher was used to punch a bacterial cake from the edge of a PDA plate of strain 3302, and strain 3302 was inoculated on PDA potato dextrose agar medium, MEA malt extract agar medium, CMD corn meal dextrose agar medium and SNA low-nutrient agar medium, respectively, with the mycelium facing down, and cultured at 25℃, 12 h light and 12 h dark conditions for 7 d, and then morphological observation was performed.

[0051] Colony morphology observation results (morphological characteristics of strain 3302 are shown in Table 1) Figure 1The colony radius was 60-61 mm on PDA after 72 h, and the dish was covered on the 4th day. The colony was ring-streaked, and conidia began to produce after 3 d, starting from the periphery of the original inoculation cake, and exuding in the aerial hyphae, which were initially white and turned green after 7 d, with a convex distribution, forming a small circular deep green lawn. The white hyphae were radial, the aerial hyphae were dense, the colony edge was distributed in an obvious band, and there was no autolysis. There was no obvious odor and secretion.

[0052] The colony radius was 72-73 mm on CMD after 72 h at 25°C. The colony was white inside and green with more sparse distribution of pustules on the outside, and was ring-shaped. There was no diffusing pigment, and no obvious odor. Conidia were produced after 3 d, and the aerial hyphae exuded and turned green after 4 d.

[0053] The colony radius was 58-59 mm on SNA after 72 h at 25°C. The colony was translucent and obvious, and the hyphae were distributed in a grid pattern, with short aerial hyphae. There was no diffusing pigment, and no obvious odor. Conidia were produced after 2 d from the periphery of the hyphal block, exuded in the aerial hyphae, and formed small pustules around the hyphal block, which were initially white and turned green after 3 d.

[0054] The colony radius was 54-56 mm on MEA after 72 h at 25°C. The colony color was from green to white, the hyphae were densely distributed, the texture was good, and the aerial hyphae were obvious. There was no diffusing pigment, and no obvious odor. Conidia were produced after 3 d, exuded in the aerial hyphae, and turned green after 4 d.

[0055] (2) Microscopic morphological observation:

[0056] The mycelium and conidia grown on PDA at 28°C for 5 d were picked up with a loop and placed on a glass slide, and one drop of 20% sterilized glycerol was added. A cover glass was placed on it, and the excess glycerol was absorbed with a water-absorbing paper. The prepared slide was observed under a Nikon biological microscope to observe the morphology of conidial phialides, phialides and conidia, and photographs were taken.

[0057] The results are shown in Figures E-N in Figure 1 The mycelium was white to translucent, branched, and 1.8-2.5 mm wide; the conidial phialides were single or whorled, tubular to phialidic, translucent, 5.51-9.45 x 2.88-4.87 μm long and wide, with an aspect ratio of 1.44-3.15, and 1.81-2.52 μm at the base; the conidia were oval to nearly spherical, smooth, transparent in youth, and green to dark green with age, 2.84-3.44 x 2.58-3.11 μm long and wide, with an aspect ratio of 0.99-1.24; and the chlamydospores were 4.52-7.72 x 4.29-7.73 μm.

[0058] 3. Molecular identification of strain 3302

[0059] The method for molecular identification of Trichoderma is to analyze the sequences of tef1-α, ITS and rpb2 genes, and the specific steps include extracting the DNA of strain 3302, PCR, sequencing and multi-gene phylogenetic tree analysis.

[0060] DNA extraction: Beijing Quanshi gold fungal genome DNA extraction kit was used for DNA extraction of Trichoderma;

[0061] PCR amplification: The primers in Table 1 were used to specifically amplify the sequences of tef1-α, ITS and rpb2 genes of strain 3302.

[0062] Sequencing: The successfully amplified gene fragments were sent to Guangzhou Tianyi Huayu Gene Technology Co., Ltd. for sequencing.

[0063] Table 1 Primers used for amplification of tef1-α, ITS and rpb2 genes of strain 3302

[0064]

[0065]

[0066] Multi-gene phylogenetic tree analysis: The reference sequences of related classification populations of Trichoderma species were obtained from NCBI GenBank, and statistical analysis of Trichoderma was performed. The concatenated dataset of tef1-α, ITS and rpb2 was used, and MAFFT was used to align each site with the sequences obtained in this study. BioEdit v 7.25 was used to check the alignment and manually adjust if necessary. Subsequently, the TrimAl tool in PhyloSuite (v1.2.2) was used to automatically trim the alignment results. Phylogenetic analysis was performed using maximum likelihood analysis (ML) in RAxML, maximum parsimony analysis (MP) in PAUP and Bayesian analysis (BP) in MrBayes (v.3.1.2). Maximum likelihood analysis was performed using RAxML-HPC2 on the CIPRES Science Gateway platform of XSEDE (8.2.12). BI analysis determined the best evolutionary model for each gene using MrModeltest v.2.3. BI analysis was performed in MrBayes v.3.1.2.

[0067] The results show that from the phylogenetic analysis of tef1-α, ITS and rpb2, it is found that the phylogenetic tree of strain 3302 is Figure 2Strain 3302 formed a separate branch under the Trichoderma population and was adjacent to T. peruvianum. The homology of the tef1-a and rpb2 genes of strain 3302 and T. peruvianum was 99.83% and 99.22%, respectively. However, there was a large difference in the branch length of the evolutionary tree between strain 3302 and T. peruvianum.

[0068] In addition, it was found from the morphology that the conidia and phialides of T. peruvianum were smaller than those of strain 3302 (3.1-4.3 x 2.4-3.7 μm and 6.6-11.4 x 5.8-9.0 μm vs 2.8-3.4 x 2.6-3.1 μm and 5.5-9.4 x 2.9-4.9 μm), and the chlamydospores of T. peruvianum were more irregular than those of 3302 (4.3-9.1 x 3.5-8.2 μm vs 4.5-7.7 x 4.3-7.7 μm). In addition, the growth rate of strain 3302 was faster than that of T. peruvianum at 25°C under PDA culture conditions.

[0069] Based on the morphological and molecular identification, strain 3302 was Trichoderma, adjacent to T. peruvianum but still had obvious interspecific differences. Therefore, Trichoderma 3302 was determined to be a new species of Trichoderma sp., named Trichoderma azadirachtae 3302, and was preserved in the China General Microbiological Culture Collection Center on May 6, 2024, with the strain preservation number CGMCC No. 3.27235.

[0070] Example 2 Evaluation of the antagonistic ability of Trichoderma azadirachtae 3302

[0071] The antagonistic ability of Trichoderma azadirachtae 3302 against the pathogenic fungi Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4) and Sclerotium rolfsii Sacc was determined by the confrontation culture method. The FocTR4 and S. rolfsii were isolated and identified from the Plant Pathology Key Laboratory of Zhongkai Agricultural Engineering College in Guangzhou, Guangdong Province, and the preserved strains were used. The specific operation is as follows:

[0072] Trichoderma azadirachtae 3302 and the two pathogenic fungi were cultured on PDA plates at 28°C for 5 days.

[0073] Then, 5mm diameter plugs were taken from the edge of the colony of T. arjanii 3302 and two pathogenic fungi, and the two pathogenic fungi and T. arjanii 3302 were inoculated into two ends of a 90mm PDA plate, respectively, with PDA plates inoculated with F. oxysporum f. sp. cubense race 4 and S. rolfsii alone as controls.

[0074] After the inoculation, the PDA plates were incubated at 25°C for 10 days, and the radius of the pathogenic fungus (R1) and the distance between the pathogenic fungus and the Trichoderma strain (R2) were measured, and the inhibition rate was calculated according to the formula: inhibition rate (%) = (R2-R1) / R2*100%, and the test was repeated three times.

[0075] The contact between the pathogenic fungus and the Trichoderma strain on the front side of the culture plate after 10 days of incubation was observed, and the hyperparasitic ability of T. arjanii 3302 was evaluated, and the evaluation grading standard is shown in Table 2:

[0076] Table 2 Evaluation grading standard for the hyperparasitic ability of Trichoderma

[0077]

[0078] The antagonistic effect of T. arjanii 3302 on F. oxysporum f. sp. cubense race 4 and S. rolfsii is shown in Figure 3 :

[0079] (1) The inhibition rate of T. arjanii 3302 on F. oxysporum f. sp. cubense race 4 was 76.85%, and the parasitic ability was complete coverage.

[0080] (2) The inhibition rate of T. arjanii 3302 on S. rolfsii was 75.09%, and the parasitic ability was moderate coverage.

[0081] Therefore, T. arjanii 3302 has strong inhibition effect on the pathogenic fungi F. oxysporum f. sp. cubense and S. rolfsii, and can be used for the prevention and control of the pathogenic fungi F. oxysporum f. sp. cubense and S. rolfsii and their diseases.

[0082] Example 3 Evaluation of the salt tolerance of T. arjanii 3302

[0083] A puncher was used to take a 5mm diameter plug of T. arjanii 3302 from the edge of the colony, and was inoculated onto PDA plates with NaCl concentrations of 0, 10, 20, 30, 40, 50, 60, and 70g / L, respectively, with a PDA plate with a NaCl concentration of 0g / L as a control (CK), and was incubated in a 28°C constant temperature incubator, and the growth diameter of T. arjanii 3302 was measured after 48h and 120h (see Figure 4 ), and the test was repeated three times.

[0084] Results: From Table 3 and Figure 4It can be seen that NaCl has a certain inhibitory effect on the growth of T. arjanicarum 3302, but with the extension of culture time, the inhibitory effect of different concentrations of NaCl on the growth of T. arjanicarum 3302 shows a decreasing trend. When the concentration of NaCl is 70 g / L, T. arjanicarum 3302 can still grow well, showing strong salt tolerance.

[0085] Table 3 Growth diameter of T. arjanicarum 3302 colony under salt stress (average value ± standard deviation)

[0086]

[0087] Example 4 Evaluation of the growth promoting ability of T. arjanicarum 3302

[0088] 1. Preparation of T. arjanicarum 3302 spore suspension and fermentation broth

[0089] Preparation of spore suspension: After T. arjanicarum 3302 was cultured on PDA solid medium for 7 days, the trichoderma spores were washed off from the medium plate under sterile operation, and the bacterial cells were filtered through 4 layers of sterile gauze. The number of trichoderma spores was calculated using a hemocytometer, and the concentration was adjusted with sterile water to obtain a spore suspension with a final concentration of 1 x 10 6 spores / mL.

[0090] Preparation of bacterial suspension: After T. arjanicarum 3302 was activated, it was transferred to PD liquid medium (formula: 200 g peeled potatoes were boiled for 30 min, then filtered, and the filtrate was obtained. After 18 g of glucose was added, distilled water was added to make the volume reach 1 L), and cultured at 28°C, 180 r / min for 3 days. Under sterile operation, the filtrate was removed by filtering through 4 layers of sterile gauze, and the water in the bacterial cells was drained. Then, 10 g of bacterial cells were put into a sterile pulverizer, 200 mL of sterile water was added, and the bacterial cells were pulverized uniformly. Finally, the bacterial suspension was diluted with sterile water to 500 mL to obtain a bacterial suspension with a final concentration of 2%.

[0091] Preparation of fermentation broth and sterile fermentation broth: After T. arjanicarum 3302 was activated, it was transferred to PD liquid medium and cultured at 28°C, 180 r / min for 3 days. After the bacterial cells were filtered out through 4 layers of sterile gauze, they were centrifuged at 3000 rpm / min for 10 min, and the supernatant was collected as the fermentation stock solution. After the fermentation stock solution was diluted by 10 times and 100 times, 10% fermentation broth and 1% fermentation broth were obtained, respectively. In addition, the fermentation stock solution was filtered through a 0.22 μm microporous filter to obtain a sterile fermentation stock solution. After the sterile fermentation stock solution was diluted by 10 times and 100 times, 10% sterile fermentation broth and 1% sterile fermentation broth were obtained.

[0092] 2. Determination of the growth promoting ability of T. arjanicarum 3302 on rice seeds

[0093] Select plump rice seeds, disinfect the seed surface by soaking in 75% alcohol for 1 minute, rinse three times with sterile water and air dry. Then soak the seeds separately in 1×10 of Trichoderma neem 3302. 6 Seeds were soaked in spore suspension, fermentation stock solution, 10% fermentation broth, 1% fermentation broth, 10% sterile fermentation broth, and 1% sterile fermentation broth for 2 hours. Seeds soaked in sterile water served as a control group. After soaking for 2 hours, the seeds were rinsed with sterile water and placed in petri dishes with moistened filter paper at the bottom and surface. Seed germination was carried out at 28℃. After 3 days, the root length and shoot length of the rice seeds were measured, and the seed germination rate was calculated using the following formula: Seed germination rate (%) = Number of germinated seeds / Total number of seeds in the experiment × 100. Each treatment contained 20 seeds, and the experiment was repeated three times.

[0094] Results: From Table 4 and Figure 5 It can be seen that *Trichoderma neem* 3302 promotes the germination of rice seeds. The 1×10⁻⁶ spores of strain 3302... 6 Spore suspension, fermentation stock solution, 10% fermentation broth, 1% fermentation broth, 10% sterile fermentation broth, and 1% sterile fermentation broth all promoted the germination rate, root length, and shoot length of rice seeds to varying degrees. In particular, the 10% fermentation broth, 10% sterile fermentation broth, and 1% sterile fermentation broth significantly promoted the germination rate, root length, and shoot length of rice seeds.

[0095] Table 4. Growth-promoting effect of Trichoderma neem 3302 on rice seeds (mean ± standard deviation)

[0096]

[0097] Note: Different letters indicate significant differences between columns, as determined by Duncan's analysis of variance using multiple comparison tests (P≤0.05).

[0098] 3. Determination of the growth-promoting ability of Trichoderma neem 3302 on banana seedlings

[0099] Select Brazilian banana seedlings (approximately 1 month old) with uniform growth and transplant them into flowerpots one week in advance. A control group (CK) was prepared by watering each banana seedling with 50 mL of sterile water, while other groups were treated with 50 mL of either a 10% fermentation broth of *Trichoderma neem* 3302, a 10% sterile fermentation broth, or a 10%... 6 Spore suspension and 2% bacterial suspension were used as treatment groups, with 10 banana seedlings in each treatment. After 40 days, root length, number of roots, stem diameter, plant height, number of leaves, fresh root weight, and fresh crown weight were measured and photographed.

[0100] Results: From Tables 5 and 6 Figure 6 It can be seen that *Trichoderma neem* 3302 can promote the growth of banana seedlings. Compared with the control group (CK), the 10% fermentation broth, 10% sterile fermentation broth, and 10% of strain 3302 showed better growth.6 The spore suspension and 2% bacterial suspension had different degrees of growth promotion on banana seedlings. Among them, the 10% sterile fermentation liquid treatment group had the most significant promotion effect on the growth of banana seedlings. The fresh root weight, fresh crown weight and root length of the treated banana seedlings were increased by 154.77%, 77.31% and 31.55% compared with the control group, and the difference was significant (P<0.05).

[0101] Table 5 Growth promotion ability of T. harzianum 3302 on banana seedlings (mean ± standard deviation)

[0102]

[0103] Table 6 Growth promotion ability of T. harzianum 3302 on banana seedlings (mean ± standard deviation)

[0104]

[0105] Note: Different letters indicate significant differences between columns by Duncan's multiple comparison test (P≤0.05).

[0106] Example 5 Greenhouse pot experiment of T. harzianum 3302 for controlling banana fusarium wilt

[0107] Select banana seedlings (about 2 months old) with consistent growth, and transplant them into pots one week in advance. Irrigate each banana plant with 50 mL of sterile water as the control group (CK), and irrigate each banana plant with 50 mL of 10% fermentation liquid of T. harzianum 3302, 10% sterile fermentation liquid, 10 6 The spore suspension and 2% bacterial suspension had different degrees of growth promotion on banana seedlings. Among them, the 10% sterile fermentation liquid treatment group had the most significant promotion effect on the growth of banana seedlings. The fresh root weight, fresh crown weight and root length of the treated banana seedlings were increased by 154.77%, 77.31% and 31.55% compared with the control group, and the difference was significant (P<0.05). 6 After 5 days of irrigation, each banana plant in all groups was treated with a wounded root, and 50 mL of banana fusarium wilt (i.e., Fusarium oxysporum f. sp. cubense race 4) spore suspension with a concentration of 1×10

[0108] Table 8 Disease grading standard for potting and inoculating banana seedlings

[0109]

[0110] Disease index = [∑(number of plants at each level × corresponding level value) / (total number of plants surveyed × highest level value)] × 100 Control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100

[0111] As shown in Table 9, Figures 7-10 T. harzianum 3302 can be used to control banana fusarium wilt. The 10% fermentation liquid, 10% sterile fermentation liquid, 106 Spore suspension and 2% bacterial suspension had different degrees of control effect on banana wilt. Among them, the 10 6 Spore suspension and 2% bacterial suspension had the most significant control effect, with external control effects of 38.24% and 45.10%, and internal control effects of 36.13% and 41.94%, respectively.

[0112] Table 9 Control effect of Trichoderma harzianum 3302 on banana wilt

[0113]

[0114] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. A new type of Trichoderma neem ( Trichoderma azadirachtae )3302 was deposited on May 6, 2024 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 3.27235.

2. The culture of Trichoderma virens 3302 as claimed in claim 1, wherein, The culture is a spore suspension, a mycelium suspension and / or a fermentation broth of Trichoderma azadirachtae 3302.

3. A microbial inoculant or fertilizer, characterized in that, The Trichoderma azadirachtae 3302 of claim 1 and / or the culture of claim 2.

4. The microbial inoculant or fertilizer of claim 3, wherein The dosage form of the microbial inoculant or fertilizer is tablet, seed coating agent, dry flowable, water dispersible granule or wettable powder.

5. Use of the Trichoderma azadirachtae 3302 of claim 1 or the culture of claim 2 or the microbial inoculant or fertilizer of any one of claims 3-4 in promoting the growth of rice or banana, inhibiting Fusarium oxysporum or Sclerotium rolfsii, or preventing plant diseases caused by Fusarium oxysporum and / or Sclerotium rolfsii.

6. Use of the Trichoderma azadirachtae 3302 of claim 1 or the culture of claim 2 or the microbial inoculant or fertilizer of any one of claims 3-4 in the preparation of a product capable of promoting the growth of rice or banana, inhibiting Fusarium oxysporum or Sclerotium rolfsii, and / or preventing plant diseases caused by Fusarium oxysporum and / or Sclerotium rolfsii.

Images