Alternaria hexangularis HZ132 and application thereof

Abstract

The invention discloses a chenopodium ambrosioides seed endophytic fungus Alternaria alstroemia HZ132. The endophytic fungus Alternaria alstroemia HZ132 is preserved in Guangdong Microbial Culture Collection Center with the preservation number of GDMCC No. 66913, and the preservation number of the endophytic fungus Alternaria alstroemia HZ132 in Guangdong Microbial Culture Collection Center is GDMCC No. 66913. The strain has strong capabilities of solubilizing phosphorus, producing indoleacetic acid, producing siderophores and resisting cadmium, is inoculated with HZ132 under cadmium stress, can significantly promote plant growth and enhance cadmium resistance of plants, and has important significance in plant-microorganism combined remediation of heavy metal contaminated soil.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of microbial technology, and particularly relates to a strain of Alternaria porri (HZ132) and application thereof in promoting plant growth and remediation of heavy metal pollution. Alternaria alstroemeriae The application belongs to the field of microbial technology, and particularly relates to a strain of Alternaria porri (HZ132) and application thereof in promoting plant growth and remediation of heavy metal pollution. BACKGROUND

[0002] Heavy metal pollution is a global environmental problem. At present, the situation of heavy metal pollution in farmland soil in China is very serious, for example, the over-standard rate of soil cadmium is nearly 35%, and the over-standard rate of lead and zinc is about 15%. Soil heavy metal pollution can reduce soil fertility, weaken the ability of soil nutrient cycling and organic matter decomposition, and thus cause crop yield reduction. If high-concentration heavy metals enter the food chain through plants, they can seriously affect plant growth and animal health, further destroy the stability of the ecological system, and affect human health. Therefore, remediation of heavy metal contaminated soil and healthy growth of crops have become urgent problems to be solved.

[0003] Among the many remediation technologies, phytoremediation mainly realizes efficient removal of heavy metals in soil by adsorbing heavy metals and transporting them to the aboveground part of hyperaccumulating plants, and is concerned due to its advantages of economy, environmental protection and in-situ remediation. However, in practical application, factors such as plant species, soil pH, organic acid, soil enzyme activity, and soil microbial community can affect the remediation effect. The single use of phytoremediation to remediate heavy metal contaminated soil has problems such as long time efficiency, low efficiency, and limited application. Therefore, improving the efficiency of phytoremediation has become a research focus. Microorganisms can colonize in plants, and promote plant growth through various ways such as biological nitrogen fixation, phosphorus solubilization, and plant hormone production, and can also enhance the resistance and enrichment capacity of plants to heavy metals. For example, Du Dongxia et al. isolated a Bacillus huizhouensis DN14 from heavy metal contaminated soil, which has the properties of cadmium solubilization, phosphorus solubilization, siderophore production, indole-3-acetic acid (IAA) production, and nitrogen fixation. Under cadmium stress, inoculation of DN14 significantly improved the growth and cadmium accumulation of the hyperaccumulating plant Solanum nigrum ( Solanum nigrum L. ). Microbial-plant combined remediation technology can effectively improve the remediation efficiency of plants to heavy metals in soil, and has good application prospect.

[0004] As a unique type of microorganism, endophytes have a more stable mutualistic symbiotic relationship with host plants, and are not easily affected by environmental factors. The beneficial effects of endophytes on hosts are usually greater than those of rhizosphere and soil microorganisms. Studies have shown that endophytes can change the absorption of plants to soil heavy metals by improving the bioavailability of heavy metals, increasing the biomass of plants, acidifying the rhizosphere environment, and increasing the root surface area, and thus play an important role in remediation of soil pollution and protection of crop growth. For example, Zhang et al. found that endophyte colonization in the hyperaccumulating plant Sedum alfredii Hance (H. ) could increase the biomass of the plant and the accumulation of cadmium in the plant, and thus improve the phytoremediation efficiency of the plant to cadmium in soil. Sedum alfrediiThe endophytic fungus *Fusarium oxysporum* (Hance) exhibits good resistance to multiple heavy metals and promotes the growth of *Sedum aizoon* and its absorption and accumulation of various heavy metals by promoting plant nutrient absorption and chlorophyll synthesis, increasing root system, and enhancing heavy metal availability. Similarly, Tovar-Sánchez et al. obtained data from legumes... Prosopis laevigata Endophytic Aspergillus thymosa C7, which exhibits significant tolerance to lead, zinc, and copper, was isolated from the roots and can enhance the translocation of heavy metals from roots to leaves in host plants. Therefore, endophytic bacteria from hyperaccumulating plants in polluted environments have broad application prospects in improving phyto-microbial remediation. Summary of the Invention

[0005] This invention provides an endophytic fungus, Alternaria hexocytoides (… Alternaria alstroemeriae HZ132, isolated from the hyperaccumulating plant *Erigeron tigrinum* (HZ132), was isolated from the plant *Erigeron tigrinum* (HZ132). Dysphania ambrosioides Seeds of L. were deposited on September 3, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 66913, 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 aforementioned Alternaria hexandrae, namely, its application in promoting plant growth and bioremediation of cadmium pollution. The endophytic fungus Alternaria hexandrae has strong phosphorus solubilization, IAA production, siderophore production, and cadmium tolerance. Under cadmium stress, it can promote the growth of host plants and enhance their cadmium resistance.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: 1. Hyperaccumulating plant *Nepeta cataria* (collected from a slag heap in a heavy metal contaminated area of ​​Ayika Village, Zhehai Town, Huize County, Qujing City, Yunnan Province) was selected. D. ambrosioides Plant samples were placed in the sun to dry until the seeds fell off naturally, and then the seed samples were stored in a refrigerator at 4°C for later use. 2. After removing the seed coat, sterilize the surface and blot dry with sterile filter paper. Randomly select surface-sterilized mature seeds and place them on a PDA plate. Incubate at 25°C and observe every other day. If fungi grow around the tissue block during incubation, remove and purify the seed. At the same time, use the washing solution test to check whether the seed surface sterilization is thorough. 3. Prepare formulations containing different amounts of Cd 2+ PDB medium of different concentrations was dispensed into Erlenmeyer flasks. Uniformly sized mycelia of the isolated endophytic strain were picked and placed into the flasks. Each concentration was repeated three times. The flasks were then incubated on a 28°C horizontal shaker at 135 rpm for 10 days. Colony growth at different concentrations was observed. The strains most resistant to Cd2+ were selected. 2+The strain with strong resistance is preserved on a PDA slant, and the fungus is named as HZ132; 4. Identification of the strain HZ132 (1) Morphological characteristics of the strain HZ132: Cultured on a PDA medium plate, the colony of the strain HZ132 is nearly round, with developed aerial hyphae, early gray, and the color of the inner ring gradually changes to brown in the later period, and the outer ring is still gray; the color of the back of the colony is deep in the middle, gradually lightens outward, and has a wheel pattern, and there are black patches distributed; (2) Molecular identification: The total DNA of the strain is extracted by using a MoBio PowerSoil ® DNA kit, and the ribosomal RNA internal transcribed spacer (ITS) gene is amplified, after detection, the sequence determination is performed by a sequencing company, the sequencing result is compared with sequences on NCBI, and an ITS sequence phylogenetic tree is constructed by using MEGA10.0 software; finally, the strain is identified as Alternaria tenuissima by combining the morphological characteristics and the molecular biological identification result; Alternaria alstroemeriae The culture medium used for preservation and activation of the strain is a PDA culture medium; 5. The endophytic fungus Alternaria tenuissima HZ132 separated from the seeds of the soil chenopod is subjected to a potting test, the influence of the endophytic fungus on the growth and cadmium stress resistance of corn is explored, the influence of inoculation of the endophytic fungus Alternaria tenuissima HZ132 on the growth of corn under cadmium stress is researched, and theoretical research basis is provided for seed endophyte-plant combined remediation.

[0008] Compared with the prior art, the present application has the following beneficial effects: The endophytic fungus Alternaria tenuissima HZ132 provided by the present application is derived from the seeds of the hyperaccumulating plant soil chenopod, has cadmium tolerance, strong phosphorus solubilization, IAA and iron carrier production capacity, and a large amount of mycelium can be obtained through simple liquid fermentation, the mycelium is easy to obtain, the cost is low, and the endophytic fungus has the potential for commercial application; inoculation of the strain HZ132 can obviously promote the growth of a host plant under cadmium stress, improve the survival ability of the host plant, and enhance the cadmium resistance of the host plant, and has important significance for remediation of heavy metal contaminated soil. DETAILED DESCRIPTION

[0009] Figure 1 It is a colony morphology of the strain HZ132 on a PDA culture medium, wherein Fig. A is a front view, and Fig. B is a back view; Figure 2 It is a phylogenetic tree of the strain HZ132; Figure 3 It is a determination result of the phosphorus solubilization capacity of the endophytic fungus Alternaria tenuissima HZ132; Figure 4 Results of IAA production capacity assay for Alternaria hexafolia HZ132; Figure 5 The results show the effects of inoculated (E+) and uninoculated (E-) Alternaria hexaflora HZ132 on maize growth under cadmium 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; 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.

[0011] Example 1: Isolation, screening and identification of the endophytic fungus Alternaria hexocytoides HZ132 (1) Hyperaccumulating plant *Nepeta cataria* collected from a slag heap in a heavy metal contaminated area in Ayika Village, Zhehai Town, Huize County, Qujing City, Yunnan Province. D. ambrosioides The plant samples were placed in the sun to dry until the seeds could fall off naturally, and then the seed samples were stored in a refrigerator at 4°C for later use. (2) After the seeds are dehulled, they are washed with tap water and then surface disinfected according to the following procedure: rinsed with 75% ethanol for 2 minutes, then rinsed with sterile water 5 times, and the above steps are repeated once. The seeds are then placed on sterile filter paper to absorb the moisture. After that, 6 mature seeds after surface disinfection are randomly selected and placed on PDA plates (90 mm) and cultured at 25°C for 60 days. The seeds are observed every other day. If fungi grow around the tissue block during the culture period, the seeds are picked and purified. At the same time, the thoroughness of seed surface disinfection is checked by the rinsing solution test. (3) Screening of cadmium-resistant strains: Preparation of Cd 2+PDB medium with concentrations of 0, 100 and 200 mg / L was dispensed into 100 mL conical flasks, and the volume of each dispensing was 50 mL. The isolated endophytic strains were inoculated into the conical flasks with uniform mycelium, and each concentration was repeated three times. The inoculated conical flasks were placed in a constant temperature horizontal shaker at 28°C and shaken at 135 rpm for 10 days. The growth of the colonies under different concentrations was observed. The strain with strong resistance to Cd 2+ was selected and preserved on a PDA slant, and the strain was named HZ132. (4) Identification of the strain HZ132 ① Morphological characteristics of the strain HZ132: The strain was cultured on a PDA medium plate, and the colony morphology was observed. The colony of the strain HZ132 was nearly round, and the aerial mycelium was developed. The early stage was grayish white, and the inner circle gradually turned brown in the later stage, while the outer circle remained grayish white. The color of the back of the colony was dark in the middle, gradually lightened outward, and had a wheel pattern with black patches Figure 1 ). ② Molecular identification: The total DNA of the strain was extracted by using a MoBio PowerSoil® DNA kit, and the ITS gene was amplified. After detection, the sequence determination company was sent for sequence determination, and the sequencing result is shown in SEQ ID NO: 1. The sequencing result was compared with the sequence on NCBI, and it was found that the strain HZ132 was clustered with Alternaria porri Alternaria alstroemeriae . Combined with the morphological and molecular biological identification results, it was clear that the strain HZ132 was Alternaria porri Alternaria alstroemeriae ), and the ITS phylogenetic tree is shown in Figure 2 .

[0012] Example 2: Cadmium resistance experiment of the endophytic fungus Alternaria porri HZ132 The isolated endophytic strains were inoculated on a PDA plate, and Cd 2+ PDB medium with concentrations of 0, 50, 100, 200, 400 and 600 mg / L was dispensed into 100 mL conical flasks, and the volume of each dispensing was 50 mL. The strain with good growth was inoculated into the conical flasks with uniform mycelium, and each concentration was repeated three times. The inoculated conical flasks were placed in a constant temperature horizontal shaker at 28°C and shaken at 135 rpm for 10 days. The growth of the mycelium was observed, and the dry weight of the mycelium was determined by the weight method. The culture solution with a Cd 2+ concentration of 0 mg / L was used as a control group, and the inhibition efficiency of the heavy metal on the strain was calculated according to the formula. Growth inhibition rate = (dry weight of control mycelium - dry weight of treated mycelium) / dry weight of control mycelium × 100% The results are shown in Table 1. Strain HZ132 can grow in PDB medium with a higher concentration of cadmium, indicating that Alternaria alternata HZ132 has good cadmium tolerance. Table 1. Cadmium tolerance of strain HZ132

[0013] Note: In the above table, "-" indicates no data, i.e. the strain cannot grow at this concentration.

[0014] Example 3: Determination of the phosphorus solubilizing ability of Alternaria alternata HZ132 After culturing Alternaria alternata HZ132 in PDA medium at 28°C for 7 days, it was inoculated into NBRIP liquid medium, and un-inoculated NBRIP liquid medium was used as a blank control. Each treatment had three replicates, and the culture was carried out at 28°C, 125 rpm on a shaker for 10 days. The soluble phosphorus content was determined by the molybdenum-antimony anti-colorimetric method (spectrophotometer wavelength 700 nm), and the specific steps were as follows: ① Centrifuge the fermentation broth at 10000 rpm for 15 min, take 1 mL of supernatant and place it in a 50 mL colorimetric tube with a stopper, add 2, 6-dinitrophenol indicator 2 drops, adjust the pH to just slightly yellow with 10% sodium hydroxide or 5% dilute sulfuric acid, add 5 mL of molybdenum-antimony color developing agent, and then add deionized water to 50 mL. ② After standing for 30 min, colorimetric determination was performed at 700 nm on a spectrophotometer, and the blank control group was also determined. ③ At the same time, a phosphorus standard curve was drawn. 0, 2, 3, 4, 5, 6, 10 mL of 5 mg / L phosphorus standard solution was taken into a 50 mL colorimetric tube with a stopper, 2 drops of 2, 6-dinitrophenol indicator was added, the pH was adjusted to just slightly yellow with 10% sodium hydroxide or 5% dilute sulfuric acid, 5 mL of molybdenum-antimony color developing agent was added, and then deionized water was added to 50 mL. 0, 0.2, 0.3, 0.4, 0.5, 0.6, 1.0 mg / L phosphorus standard series solutions were obtained. After standing for 30 min, colorimetric determination was performed at 700 nm on a spectrophotometer, and a standard working curve y=0.52x+0.0028 (R 2 =0.9998) was drawn. Then the absorbance value measured in step ② was substituted into the standard working curve, and the phosphorus solubilizing amount of Alternaria alternata HZ132 in NBRIP culture solution was obtained as 100.468 mg / L Figure 3 ), indicating that Alternaria alternata HZ132 has strong phosphorus solubilizing ability and can convert insoluble inorganic phosphorus into soluble phosphorus, making it easier for plants to absorb phosphorus and promoting plant growth, especially in areas with heavy metal pollution leading to nutrient-poor growth.

[0015] Example 4: Determination of the ability of Alternaria alternata HZ132 to produce IAA Alternaria alternata HZ132 was cultured in PDB medium (containing L-tryptophan 0.5 mg / mL) at 28℃, 125 rpm for 10 days. 1 mL of bacterial solution was centrifuged at 12000 rpm for 15 min, and the precipitate was removed. 1 mL of supernatant was added with an equal amount of Salkowski's reaction solution (1 mL of 0.5 mol / L ferric chloride, 49 mL of 35% perchloric acid), and reacted in the dark for 30 min. The absorbance value was measured at 530 nm. Each group was repeated three times, and the uninfected medium was treated as above to set zero. The absorbance value of the IAA standard solution with concentrations of 0, 2.5, 5, 10, 20, 40, 60, 80, 100 mg / L was determined by the above method, and the standard curve y=0.0244x+0.0148 (R 2 =0.9982) was obtained. Then the absorbance value of the experimental group inoculated with Alternaria alternata HZ132 was substituted into the standard curve, and the IAA yield of Alternaria alternata HZ132 was 23.432 mg / L (S Figure 4 ), Alternaria alternata HZ132 had strong ability to synthesize plant hormone IAA, and had the potential to promote plant cell growth and proliferation, and regulate host heavy metal absorption and transport.

[0016] Example 5: Determination of the ability of endophytic fungus Alternaria alternata HZ132 to produce siderophores Alternaria alternata HZ132 was cultured in PDA medium at 28℃ for 7 days, and then uniform mycelial blocks were inoculated into iron-free Czapek's medium (sucrose 30 g, sodium nitrate 2 g, potassium phosphate dibasic 1 g, magnesium sulfate heptahydrate 0.5 g, potassium chloride 0.5 g, distilled water 1 L). After 5 days of shaking culture at 28℃, 125 rpm, the culture supernatant was collected by centrifugation at 8000 rpm for 10 min. 1 mL of supernatant was mixed with 1 mL of CAS detection solution, and the mixture was reacted in the dark for 15 min. The absorbance value was measured at 630 nm, which was denoted as As. The OD 630 value was measured in the same way in the blank control, which was denoted as Ar. Deionized water was used as a control to set zero. The specific calculation formula was siderophore activity=[(Ar-As) / Ar]x100%. The calculation result was that the siderophore activity of strain HZ132 was 51.15%. The secretion of siderophores enables microorganisms and plants to capture the iron elements required for life activities. In addition, siderophores have many applications in the fields of environmental pollution and plant growth promotion, such as accelerating mineral dissolution, promoting soil heavy metal and organic pollutant remediation, and affecting plant growth and biological control, etc.

[0017] Example 6: Effects of the endophytic fungus Alternaria hexocytoides HZ132 on maize growth under heavy metal stress This embodiment aims to demonstrate the promoting effect of Alternaria hexafolia HZ132 on plant growth and heavy metal tolerance in heavy metal pollution; using maize ( Zea mays L.) was the test plant, and the experimental procedure was as follows: Preparation of Alternaria hexafolia HZ132 suspension: The previously purified HZ132 strain was selected, inoculated into PDA plates for activation, and cultured in a water-jacketed incubator at 28℃ for 7 days. Then, the mycelium was picked from the plates with good growth and no contamination and inoculated into PDB medium. After culturing in a constant temperature shaker at 28℃ and 125 rpm for 7 days, the mycelium was filtered out under aseptic conditions and rinsed 5 times with sterile water to avoid the mycelium from being contaminated with medium. After being thoroughly squeezed dry, the mycelium was cut into small pieces with sterile scissors. The cut mycelium was then diluted with sterile water to make a 5% HZ132 mycelial suspension. Seed soaking method for inoculation with Alternaria hexandrum HZ132 and maize germination: Randomly selected maize seeds were first soaked in a 75% ethanol solution for 2 minutes, rinsed 3 times with sterile water, then soaked in a 5% NaClO solution for 1 minute, rinsed 5 times with sterile water, and then blotted dry on sterile filter paper for surface sterilization. The surface-sterilized maize seeds were then soaked in the above HZ132 bacterial suspension (E+) for 24 hours for colonization. Seeds soaked in an equal volume of sterile water served as the control group (E-). The soaked seeds were then placed in soil that had been autoclaved (124℃, 30 minutes) and germinated at room temperature (18-25℃) and 60% relative humidity. The soil formula was V... 田园土 V 加拿大泥炭藓 V 珍珠岩 =7:2:1, and to maintain moisture, water with sterile water every 3 days.

[0018] Pot experiment: Seven days after corn seed germination, 8 seedlings of uniform growth were selected from each group (E+ / E-) and transplanted into flowerpots (14.5×16.5cm, 1 seedling / pot). Each pot contained 1.5kg of autoclaved (124℃, 30min) substrate (V). 田园土 :V 加拿大泥炭藓 V 珍珠岩 =7:2:1, Contains 15mg / L Cd 2+) ; the seedlings were placed in a room temperature with 14 / 10h light and dark cycle illumination culture, and irrigated with sterile water every 2-3 days, 50mL per pot (it is appropriate that the water irrigates the soil thoroughly without overflowing the bottom of the pot) ; after 7 days of transplantation, 10mL of 5% concentration of Alternaria alternata HZ132 bacterial suspension was irrigated on the roots of the plants in the E+ group, and the same amount of sterile water was irrigated on the roots of the plants in the E- group; the growth status of the corn was observed closely during the potting process, and the corn was harvested after 30 days, and the plant height, root length, and dry weight of the aboveground and underground parts of the corn were measured.

[0019] The results are shown in Table 1. Figure 5 As shown in Table 1, under the stress of cadmium, the growth-promoting effect of inoculating Alternaria alternata HZ132 on the corn seedlings was obvious, and the root length and underground dry weight of the corn in the E+ group were significantly higher than those in the E- group (p<0.05, t test), indicating that the endophytic fungus Alternaria alternata HZ132 can effectively promote the growth of the host plant in the heavy metal polluted environment and enhance the heavy metal resistance of the host plant, and has good growth-promoting effect and heavy metal remediation potential.

[0020] The results of the above examples show that the endophytic fungus Alternaria alternata HZ132 isolated and obtained in the present application has strong abilities of phosphorus solubilization, IAA and siderophore production, has excellent cadmium tolerance, and has good growth-promoting effect on the corn seedlings and enhances the heavy metal resistance of the corn seedlings under the stress of heavy metal.

Claims

1. A strain of Alternaria hexalidosa ( Alternaria alstroemeriae HZ132, its accession number at the Guangdong Provincial Center for Microbial Culture Collection is GDMCC No. 66913.

2. The application of Alternaria hexafolia HZ132 as described in claim 1 in promoting plant growth and bioremediation of heavy metal cadmium pollution.

3. The application according to claim 2, characterized in that: Alternaria hexafolia HZ132 has the ability to solubilize phosphorus, produce indoleacetic acid and iron carriers, and is cadmium resistant.

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