Talaromyces sp. d117 in seeds of upland rice and application thereof

Abstract

The application discloses a strain of Talaromyces intermedial (D117) inbred in seeds of upland rice, Diaporthe tulliensis The strain has strong abilities of producing indole acetic acid (IAA), dissolving phosphorus and producing iron carrier, can be inbred in rice, can significantly enhance the drought resistance of the rice, and can significantly improve the root length, fresh weight, overground dry weight and underground dry weight of crops, and provides excellent strain resources for development of new microbial fertilizers.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to an endophytic *Metacarbamoides tamariscina* strain isolated from upland rice seeds in Luoping County, Qujing City, Yunnan Province. Diaporthe tulliensis D117 has the ability to produce indoleacetic acid (IAA), lyse phosphorus, and produce iron carriers; it can be used to enhance the drought resistance of crops. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is one of the world's major food crops, with approximately 160 million hectares of land on Earth used for rice cultivation, mostly in Asia, representing about 46.6% of the world's inhabited area. However, global climate change is exacerbating the frequency, intensity, and duration of droughts, making water scarcity a serious problem and posing an unprecedented threat to agricultural production. As a water-intensive crop, rice production is particularly severely affected, consuming approximately 69% of global agricultural water. Currently, more than one-third of the world's cultivated rice is affected by drought stress, with 33% in developing countries, 25% in developed countries, and 42% in less developed countries. As one of my country's major food crops, rice accounts for 29.1% of the total crop planting area and 43.7% of my country's total grain output. Meta-analysis by Mao Zilin et al. shows that the average yield reduction of rice under drought is 24.0%, with the largest yield reduction (24.4%) occurring under drought stress during the vegetative growth stage. Therefore, stable rice production is of great significance to my country's food security. Traditional research on improving plant drought resistance mainly involves molecular marker-assisted breeding and genetic engineering, but these methods are highly labor-intensive and technical, and therefore difficult to apply comprehensively to agricultural practices at present.

[0003] Endophytic microorganisms are microorganisms that exist inside plants and do not cause obvious infection symptoms in the host plant. Utilizing plant symbiotic microorganisms is one of the potential strategies to improve crop drought resistance. Current research reports that some fungi (such as arbuscular mycorrhizal fungi) have the ability to promote plant metabolism and development by promoting plant water absorption through their extensive hyphae. In addition, some rhizosphere growth-promoting bacteria can also help plants resist drought stress through multiple mechanisms. Another strategy for plants to survive under drought conditions is to use growth-promoting bacteria and fungi. These microorganisms can inhibit or alleviate certain plant diseases and mobilize some micronutrients to promote the growth of the host plant.

[0004] Existing strains generally suffer from problems such as limited functionality, poor environmental adaptability, and unstable growth-promoting effects. In particular, strains that can adapt to the arid and barren conditions of the Yunnan Plateau and have a specific growth-promoting effect on rice are even rarer. Therefore, isolating and identifying highly drought-resistant endophytic fungal strains and developing their applications in agriculture has significant scientific value and promising application prospects. Summary of the Invention

[0005] To address the problems of limited functionality, insufficient environmental adaptability, and especially the lack of drought-resistant and growth-promoting strains for crops in existing technologies, this invention provides an endophytic *Talimella tamariscina* strain for upland rice seeds. Diaporthe tulliensis D117, isolated from dryland rice seeds from Luoping County, Qujing City, Yunnan Province, was deposited on January 21, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 67719, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] Another objective of this invention is to provide a new use for the above-mentioned endophytic *Taliscarpus tamariscina* D117 in upland rice seeds, namely, to enhance the drought resistance of rice. The strain D117 of this invention has the ability to produce indoleacetic acid (IAA), phosphate solubilizers and siderophores, and has a significant promoting effect on rice growth.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: 1. Collect dryland rice seed samples from Luoping County, Qujing City, Yunnan Province, and rinse them clean under tap water; 2. Surface sterilize the dryland rice seed samples, and place the surface-sterilized seeds on sterile filter paper to absorb moisture. Use the tissue block isolation method to isolate fungi. Cut the sterilized dryland rice seeds with sterile scissors and forceps, and randomly select 100 tissue blocks. Place them in PDA plates, press them to adhere to the culture medium, with 4 tissue blocks per plate, for a total of 25 plates. Incubate at room temperature for 28 days, observing them at any time. If hyphae grow, mark them immediately, isolate and purify them into PDA slant for preservation. 3. The ability of the isolated endophytic fungal strains to produce indoleacetic acid was determined. 4. Select strains with strong indoleacetic acid production capacity and preserve them on PDA slant for later use. In this experiment, a strain with good comprehensive three functions was screened and obtained, and the fungal strain was named D117. 5. Identification of strain D117 ①D117 morphological characteristics: The colonies grow relatively quickly on PDA medium. In the early stage, the hyphae are grayish-white with dense aerial hyphae that creep on the surface of the medium. In the later stage, they turn grayish-brown. ② Molecular identification: Total DNA was extracted from this strain using the MoBio PowerSoil® DNA kit, and the internal transcribed spacer (ITS) gene of ribosomal RNA was amplified. After detection, the DNA was sent to Sangon Biotech for sequencing. The sequencing results were compared with sequences in the NCBI database, and a phylogenetic tree of the ITS sequence was constructed using MEGA10.0 software. Combining morphological characteristics and molecular biological identification results, this strain was finally identified as *Talimella tamariscina*. Diaporthe tulliensis The culture medium used for the preservation and activation of this strain was PDA medium. 6. This invention conducts pot experiments on the endophytic *Talimella tamariscina* D117 isolated from dry rice to explore its effect on rice seedling growth under drought stress. In other words, it studies the effect of *Talimella tamariscina* D117 inoculation on potted rice growth, providing fungal strains and theoretical research basis for microbial fertilizers.

[0008] Compared with the prior art, the present invention has the following beneficial effects: The endophytic *Metacarba tamariscina* D117 provided by this invention has the ability to produce IAA, solubilize phosphorus, and produce siderophores. Moreover, a large amount of mycelium can be obtained through simple liquid fermentation. The mycelium is easy to obtain and inexpensive, and has the potential for commercial application. Inoculating with strain D117 can significantly promote the growth of host plants under drought stress and increase the biomass of crops under drought stress, which is of great significance for the stress resistance of crops. Attached Figure Description

[0009] Figure 1 The colony morphology of strain D117 on both sides of PDA medium; Figure 2 Phylogenetic tree of strain D117; Figure 3 This is the standard working curve for IAA standard solutions; Figure 4 The results show the assay results of the IAA secretion capacity of strain D117; Figure 5 The standard working curve for phosphorus standard solution; Figure 6 The results are for the phosphorus solubility assay of strain D117; Figure 7 The effects of strain D117 on the growth of rice seedlings under drought stress. Detailed Implementation

[0010] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the present invention is not limited to the following technical solutions. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Those skilled in the art can implement the invention by referring to various commonly used reference books, scientific and technological documents, or related instructions and manuals prior to the filing date of this invention.

[0011] The preparation of the chromium azurite S (CAS) detection solution in the following examples is as follows: Solution A: Weigh 60.5 mg of chromaine and dissolve it in 50 mL of deionized water, then add 10 mL of Fe. 3+ The solution (containing 1 mmol / L ferric chloride hexahydrate and 10 mmol / L dilute hydrochloric acid) was stirred and mixed thoroughly. Solution B: Dissolve 72.9 mg of cetyltrimethylammonium bromide in 40 mL of deionized water; Slowly pour solution A into solution B and stir until homogeneous to obtain the CAS blue detection solution.

[0012] Example 1: Isolation, screening and identification of strain D117 (1) Collect seeds of Yunlu 142 dryland rice at maturity in Luoping County, Qujing City, Yunnan Province, and rinse them clean under tap water; (2) After the dryland rice seed samples were dehulled, they were surface disinfected. First, they were soaked in 75% ethanol solution for 2 minutes, rinsed 3 times with sterile water, and then soaked in 5% sodium hypochlorite solution for 1 minute, rinsed 5 times with sterile water. After rinsing, they were placed on sterile filter paper to absorb the water. Fungal isolation was carried out using the tissue block isolation method. The disinfected dryland rice seeds were cut with sterile scissors and tweezers, and 100 tissue blocks were randomly selected and placed in PDA plates (90 mm). They were pressed to adhere to the culture medium. There were 4 tissue blocks on each plate, and 25 plates were placed on each plate. The plates were incubated at room temperature for 28 days. During this period, the samples were observed at any time. If hyphae grew, they were marked and isolated and purified into PDA slant for preservation. At the same time, the thoroughness of seed surface disinfection was checked by the rinsing solution test. (3) After the isolated endophytic strains were cultured in PDA medium at 28°C for 7 days, 10 mm × 10 mm mycelial blocks were obtained by punching holes. One mycelial block of each strain was picked and inoculated into PDB liquid medium containing 0.5 mg / mL L-tryptophan (200 g potato, 20 g glucose, 1 L distilled water, natural pH) and cultured on a shaker at 28°C and 125 rpm for 10 days. The bacterial solution was centrifuged at 12000 rpm for 10 min, the precipitate was removed, and 1 mL of the supernatant was added to an equal volume of Salkowski's reaction solution (2 mL 0.5 mol / L ferric chloride, 98 mL 35% perchloric acid). The reaction was carried out in the dark for 30 min. If the strain could produce IAA, the liquid would be pink after the reaction. The absorbance of the reaction solution at 530 nm was measured. Each group was repeated three times. The liquid medium without inoculation was used as a control and the above steps were performed to zero the sample. Using a 200 mg / L IAA standard solution as the stock solution, a series of IAA standard solutions with concentrations of 0, 2.5, 5, 10, 20, 40, 60, 80, and 100 mg / L were prepared by serial dilution. 1 mL of each concentration of IAA standard solution was mixed with 1 mL of Alkowski's reaction solution, and the mixture was allowed to react in the dark for 30 min. The absorbance at 530 nm was then measured, and a standard curve was plotted: y = 0.0221x + 0.0315 (R²). 2 =0.9927)( Figure 3 Then, the absorbance values ​​of the tested strains were substituted into the standard curve to obtain the IAA production capacity of the strains. The indoleacetic acid production capacity of the strains was quantitatively determined using Salkowski's reaction solution. Strains with strong indoleacetic acid production capacity were selected and preserved on PDA slant for later use. Among the isolated endophytic strains, strain D117 had an IAA production of 30.12 mg / L. Figure 4 ); (4) Identification of strain D117 ①Morphological characteristics of D117: When cultured on PDA agar plates, the colony morphology was observed. This strain has a relatively fast growth rate; initially, the hyphae are white with dense aerial hyphae that creep along the surface of the PDA agar plate, gradually turning grayish-brown in the later stages. Figure 1 ); ② Molecular identification: Total DNA was extracted from this strain using the MoBio PowerSoil® DNA kit, and the ITS gene fragment was amplified. After electrophoresis detection, the DNA was sent to a sequencing company for sequencing. The sequencing results are shown in SEQ ID NO:1. The sequencing results were compared with the sequence on NCBI. Diaporthe tulliensis Homology reached 99.66%; Apiognomonia pseudohystrix As an outgroup, the phylogenetic tree constructed based on ITS shows that the tested strain D117 is related to... Diaporthe tulliensisThe reference strains clustered together; combining morphological characteristics and molecular identification results, strain D117 was finally identified as... Diaporthe tulliensis ITS phylogenetic tree as follows Figure 2 As shown.

[0013] Example 2: Determination of the phosphorus-solubilizing ability of endophytic *Metacarbamochaeta* D117 Endophytic *Metacarba tamariscina* D117 was cultured in PDA medium at 28°C for 7 days. 10mm × 10mm mycelial blocks were obtained by punching holes in the medium. Three blocks were inoculated into inorganic phosphorus liquid medium (10g glucose, 5g magnesium chloride hexahydrate, 0.2g potassium chloride, 0.25g magnesium sulfate heptahydrate, 5g tricalcium phosphate, 0.1g ammonium sulfate, 1L distilled water, pH 7.0). An uninoculated inorganic phosphorus liquid medium served as a blank control. Each treatment was repeated in triplicate. The culture was carried out at 28°C and 125 rpm on a shaker for 10 days. The soluble phosphorus content was determined using the molybdenum antimony colorimetric method (spectrometer wavelength 700nm). The specific steps are as follows: ① Centrifuge the fermentation broth at 10,000 rpm for 15 minutes, take 1 mL of the supernatant and put it into a 50 mL stoppered colorimetric tube, add 2 drops of 2,6-dinitrophenol indicator, adjust the pH with 10% sodium hydroxide or 5% dilute sulfuric acid until the solution is just slightly yellow, add 5 mL of molybdenum anti-color development agent and then add deionized water to make up to 50 mL. ②After standing for 30 minutes, the colorimetric sample was measured at 700 nm using a spectrophotometer, and the blank control group was measured at the same time. ③ Simultaneously, a phosphorus standard curve was plotted. 0, 2, 4, 6, 8, and 10 mL of 5 mg / L phosphorus standard solution were respectively pipetted into 50 mL stoppered colorimetric tubes. Two drops of 2,6-dinitrophenol indicator were added. The pH was adjusted with 10% sodium hydroxide or 5% dilute sulfuric acid until the solution was just slightly yellow. 5 mL of molybdenum antimony colorimetric reagent was added, followed by dilution with deionized water to obtain a series of phosphorus standard solutions of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / L. After standing for 30 min, the solutions were measured at 700 nm using a spectrophotometer. The standard working curve y = 0.5271x (R0) was plotted. 2 =0.9981)( Figure 5 Then, the absorbance value measured in step ② was substituted into the standard working curve to obtain the phosphorus solubility of endophytic *Tallisneria natans* D117 in inorganic phosphorus culture medium as 23.3 mg / L. Figure 6 This indicates that it has the ability to convert insoluble inorganic phosphorus into soluble organic phosphorus, making it easier for plants to absorb phosphorus and thus promoting plant growth.

[0014] Example 3: Determination of the siderophore production capacity of endophytic *Metacarbamate* D117 Endophytic *Tamarix tali* D117 was cultured in PDA medium at 28°C for 7 days. Mycelial blocks of 10 mm × 10 mm were obtained by punching holes. One mycelial block was inoculated into iron-free Czapek's medium (30 g sucrose, 2 g sodium nitrate, 1 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.5 g potassium chloride, and 1 L distilled water). After incubation at 28°C and 125 rpm with shaking for 5 days, the culture supernatant was collected after centrifugation at 8000 rpm for 10 min. 1 mL of the supernatant was mixed with 1 mL of CAS detection solution, and the mixture was allowed to react in the dark for 15 min. The absorbance was measured at 630 nm and recorded as As. The OD value was measured in the blank control using the same method. 630 The value, denoted as Ar, was zeroed using deionized water as a control. The specific calculation formula is: siderophore activity = [(Ar-As) / Ar] × 100%; the calculated result shows that the siderophore activity of strain D117 is 52.77%. Siderophore secretion enables plants to capture iron elements needed for life activities, promotes plant growth, and thus improves stress resistance.

[0015] Example 4: Experiment on the growth-promoting and drought-resistance effects of endophytic *Metacarbamoides tamariscina* D117 on rice seedlings under drought stress. This embodiment aims to demonstrate the drought-promoting and drought-resistant effects of the endophytic *Metacarba talyssum* D117 on rice under drought stress. The experimental procedure is as follows: Preparation of endophytic fungal inoculum: The purified endophytic *Metacarba tamariscina* D117 was inoculated into PDA plates for activation and cultured in a 28°C water-jacketed incubator for 7 days. After that, the plates with good growth and no contamination were selected, and mycelia were picked and inoculated into PDB medium. After culturing in a 28°C, 125 rpm constant temperature shaker for 7 days, the mycelia were filtered out under aseptic conditions and rinsed 5 times with sterile water to avoid the mycelia from being contaminated with medium. After being thoroughly squeezed dry, the mycelia were cut into small pieces with sterile scissors. The cut mycelia were then diluted with sterile water to make a 5% D117 mycelial suspension. Preparation and soaking of sterile rice seeds: Plump rice seeds (Taiyou 808) were randomly selected. They were first soaked in a 75% ethanol solution for 2 minutes, rinsed three times with sterile water, then soaked in a 5% NaClO solution for 1 minute, rinsed five times with sterile water, and finally blotted dry on sterile filter paper. Half of the surface-sterilized rice seeds were soaked in the above-mentioned D117 bacterial suspension (E+) for 24 hours for colonization, while the other half were soaked in an equal volume of sterile water as the control group (CK). The soaked seeds were then shallowly buried in soil that had been autoclaved (124℃, 30 minutes) and germinated at room temperature (18-25℃) and 60% relative humidity. To maintain moisture, the seeds were watered with sterile water every two days. C. Rice seedling management: In order to maintain moisture and nutrients, water with sterile water every 2 days and apply fertilizer water with a concentration of 1g / L every 15 days. After 30 days, when the rice seedlings have grown to the 2-leaf and 1-heart stage, transplant them. D. Pot experiment: Select rice seedlings with uniform growth and transplant them into pots (1 seedling per pot, 10 pots each for the E+ group and CK group). Each pot contains 400g of farmland soil, and base fertilizer is applied in the early stage. Three days after transplanting, 10 mL of 5% endophytic *Tallisneria natans* D117 bacterial suspension was applied to the roots of E+ seedlings, while the roots of CK group seedlings were applied with an equal amount of sterile water. Thirty days after transplanting, the same bacterial suspension and sterile water were applied again. Thirty days after the second application, a 14-day drought stress was imposed, with the soil moisture content set at 45%–50% of field capacity. During this period, the potted plants were weighed and recorded daily, and water was added according to the soil moisture requirements. The growth of rice seedlings in each group was closely observed during the experiment. After the drought stress ended, the plants were harvested, and the plant height, root length, plant fresh weight, root dry weight, stem and leaf dry weight, and chlorophyll content were measured.

[0016] See results Figure 7 The results showed that the endophytic *Talimella tamariscina* D117 significantly promoted the drought resistance of rice seedlings under drought stress. After 14 days of drought stress, there were significant differences in root length, fresh weight, aboveground dry weight, and underground dry weight between the E+ and CK groups (p<0.05, t-test). Compared with CK, root length, fresh weight, aboveground dry weight, and underground dry weight increased by 14.08%, 19.68%, 32.97%, and 44.42%, respectively, indicating that the endophytic *Talimella tamariscina* D117 can effectively promote the growth of the host plant under drought stress and has a good drought resistance and growth-promoting effect.

[0017] The results of the above embodiments demonstrate that the endophytic *Tamarix talicum* D117 isolated and obtained in this invention has the ability to produce indoleacetic acid, solubilize phosphorus, and produce siderophores, thereby promoting the absorption of phosphorus and iron by plants, and also providing plant hormones to promote plant growth. This indicates that inoculation with endophytic *Tamarix talicum* D117 can promote the growth and development of rice and enhance its resistance to drought.

Claims

1. Endophytic *Talismania tamariscina* in a dryland rice seed ( Diaporthe tulliensis D117, whose accession number at the Guangdong Provincial Center for Microbial Culture Collection is GDMCC No. 67719.

2. The application of the endophytic *Taliscarpus tamariscina* D117 in dryland rice seeds as described in claim 1 in enhancing the drought resistance of rice, characterized in that: The endophytic fungus D117 in upland rice seeds has the ability to produce indoleacetic acid, phosphate solubilizers, and siderophores.

3. The application of the endophytic *Taliscarpus tamariscina* D117 in dryland rice seeds as described in claim 1 in promoting rice growth.

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