A method for promoting rice growth and inhibiting lead accumulation in rice grains by using penicillium oxalicum sl2 and application thereof
Patent Information
- Application Number
- CN202610817353.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-08
- Publication Date
- 2026-08-18
AI Technical Summary
[0006]本发明针对现有技术水稻中降铅与促生难以协同、籽粒铅转运阻控效果差、根际定殖不稳定的技术缺陷，提供一种操作简便、效果显著、机制清晰的利用草酸青霉SL2促进水稻生长并阻控籽粒铅累积的方法及应用
（1）实现降铅与促生的高效协同，保障粮食安全：本发明提供的方法在500 mg/kg铅污染土壤中应用，可使成熟期水稻籽粒铅含量从0.55 mg/kg显著降低至0.174 mg/kg，降幅达68.4%，满足《食品安全国家标准 食品中污染物限量》（GB 2762-2022）规定的0.2 mg/kg限值要求，有效保障了稻米食用安全。同时使水稻成熟期生物量提高14.2%，单株穗重增加15.0%，实现了“促生”与“降铅”双重目标，助力实现中轻度污染农田实施安全利用目标。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural biotechnology and safe utilization of lead-contaminated farmland, specifically involving a method and application of using Penicillium oxalate SL2 to promote rice growth and control lead accumulation in grains. Background Technology
[0002] Lead (Pb) is a neurotoxic, cumulative heavy metal that can accumulate through the food chain, causing irreversible damage to the human hematopoietic and nervous systems, particularly impacting children's intellectual development. According to the "National Soil Pollution Status Survey Bulletin," the lead contamination rate in farmland soil reached 1.5%, with lead pollution in paddy fields near industrial and mining areas being particularly prominent. Excessive lead in rice has become a key bottleneck restricting my country's food quality and safety. The "Soil Pollution Prevention and Control Law" explicitly requires the safe utilization of moderately and lightly polluted farmland, stipulating that "priority should be given to bioremediation measures that do not affect agricultural production or reduce soil productivity, blocking or reducing the entry of pollutants into the edible parts of crops, and ensuring the quality and safety of agricultural products." Therefore, developing low-cost, eco-friendly remediation technologies that do not affect crop yields has become an urgent need in the field of agricultural environment.
[0003] Traditional lead-contaminated soil remediation technologies, particularly physicochemical methods (such as topsoil replacement, leaching, and solidification stabilization), suffer from drawbacks such as high costs, damage to soil structure, and potential secondary pollution, making large-scale application in farmland difficult. Microbial remediation, on the other hand, has become a research hotspot for heavy metal pollution remediation in farmland due to its strong environmental compatibility, low cost, and ability to improve soil ecology. Existing research indicates that some microorganisms can alter the form of heavy metals and reduce their bioavailability through extracellular adsorption, bioprecipitation, or redox reactions; additionally, some rhizosphere growth-promoting bacteria (PGPRs) can promote crop growth by secreting plant hormones and activating soil nutrients.
[0004] However, existing technologies mostly focus on single pollution remediation or growth-promoting effects, and suffer from bottlenecks such as poor rhizosphere colonization stability. There is a lack of efficient, stable, and clearly defined technical solutions that can synergistically achieve the dual goals of "growth promotion" and "lead reduction." Most microbial strains only possess single heavy metal remediation or growth-promoting functions. While some lead-reducing strains can decrease soil lead availability, they inhibit rice root development, leading to yield reduction. Growth-promoting strains, on the other hand, have no significant inhibitory effect on lead pollution and cannot solve the problem of excessive lead levels in rice. In particular, effectively controlling the translocation of lead from the roots to edible parts such as rice grains remains a critical challenge that urgently needs to be addressed.
[0005] Therefore, screening for specialized microbial strains that possess high lead tolerance, efficient lead reduction capabilities, strong rhizosphere colonization ability, and significant growth-promoting functions, and establishing a safe cultivation technology system that can control lead translocation to rice grains from the source, is the core key to achieving safe production in rice fields with moderate to mild lead pollution, and has significant theoretical and applied value. Summary of the Invention
[0006] This invention addresses the shortcomings of existing technologies in rice, such as the difficulty in synergistically reducing lead and promoting growth, poor control of lead translocation in grains, and unstable rhizosphere colonization. It provides a simple, effective, and clearly mechanistic method and application for promoting rice growth and controlling lead accumulation in grains using Penicillium oxalate SL2.
[0007] The technical solution adopted in this invention is as follows: This invention provides a method for promoting rice growth and inhibiting lead accumulation in grains using Penicillium oxalate SL2. Specifically, during the rice seedling stage, a Penicillium oxalate SL2 spore suspension is used to co-culture and inoculate rice seedlings with the roots, allowing the strain to successfully colonize the rice rhizosphere. The treated seedlings are then transplanted into lead-contaminated soil for cultivation. The method includes the following steps: The tested strain, Penicillium oxalicum SL2, is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 2014505.
[0008] S1: Preparation of Penicillium oxalate SL2 spore suspension; S2: Immerse the roots of 14-day-old rice seedlings in the spore suspension at a depth of 4-6 cm and culture them for 7 days at 28°C under 16h light / 8h darkness conditions to colonize Penicillium oxalate SL2 in the rice rhizosphere. S3: Transplant the inoculated rice seedlings into lead-contaminated soil and cultivate them to maturity using a phased water management model.
[0009] Preferably, the preparation method of the Penicillium oxalate SL2 spore suspension described in S1 is as follows: Penicillium oxalate SL2 is inoculated into potato dextrose agar medium and cultured at 28°C for 7 days. Fresh spores are harvested, and a spore suspension stock solution is prepared using 0.01% sterile Triton X-100 solution. A working concentration of 1×10⁻⁶ is prepared using Kimura nutrient solution. 5 - 1×10 7 A spore suspension of 1 spore per mL.
[0010] Preferably, the working concentration of the spore suspension is 1×10⁻⁶. 6 per mL.
[0011] Preferably, the lead-contaminated soil in step S3 is weakly acidic farmland soil with a pH value of 5.5 to 6.5 and a total lead content of ≤500 mg / kg.
[0012] As a preferred option, the phased water management mode in step S3 is as follows: continuously flood the field for 60 days before the heading stage, maintaining a water layer of 2-3 cm in the field, and after the heading stage, use alternating flooding and drying treatment to cultivate the rice until maturity, and harvest the rice plants 100 days after transplanting.
[0013] As a preferred method, the specific approach to the alternating flooding-drying treatment is as follows: irrigate with shallow water 2-3 cm, wait for the water layer in the field to dry naturally for 1-2 days, and then re-irrigate, repeating this cycle until maturity.
[0014] This invention also provides the application of Penicillium oxalate strain SL2 in promoting rice growth and inhibiting lead accumulation in grains, wherein the Penicillium oxalate strain SL2 is... Penicillium oxalicum SL2, with accession number CCTCC NO: M2014505.
[0015] This invention also provides a microbial agent for promoting rice growth and inhibiting lead accumulation in grains, wherein the microbial agent uses *Penicillium oxalate* SL2 as the active ingredient, and the *Penicillium oxalate* SL2 strain is... Penicillium oxalicum SL2, with accession number CCTCC NO: M 2014505.
[0016] Preferably, the microbial agent is a spore suspension, and the concentration of the spore suspension is 1×10⁻⁶. 5 ~1×10 7 per mL.
[0017] Compared with the prior art, the present invention has the following advantages: (1) Achieving efficient synergy between lead reduction and growth promotion to ensure food security: The method provided by this invention, when applied to lead-contaminated soil at 500 mg / kg, can significantly reduce the lead content of mature rice grains from 0.55 mg / kg to 0.174 mg / kg, a reduction of 68.4%, meeting the 0.2 mg / kg limit requirement stipulated in the National Food Safety Standard for Maximum Levels of Contaminants in Food (GB 2762-2022), effectively ensuring the safety of rice consumption. At the same time, it increases the biomass of mature rice by 14.2% and the panicle weight per plant by 15.0%, achieving the dual goals of "growth promotion" and "lead reduction," and helping to achieve the goal of safe utilization of moderately and lightly polluted farmland.
[0018] (2) Solving the problem of unstable rhizosphere colonization of functional strains: The inoculation method of root co-culture during the seedling stage was adopted, and the optimized parameters of 5 cm immersion height were used to make Penicillium oxalate SL2 stably colonize in the iron film on the root surface of rice.
[0019] (3) The dual lead-blocking mechanism is clear: The mechanism of action of the present invention is clear and reliable. Penicillium oxalate SL2 blocks the absorption and transport of lead through the dual action of strengthening the iron membrane interception on the root surface and enhancing the fixation of the root cell wall: the proportion of lead in the iron membrane on the root surface increases from 6.52% to 11.27%, and the proportion of lead in the root cell wall increases from 39.49% to 52.48%, thus preventing lead from entering rice cells and being transported to the aboveground parts at the source.
[0020] (4) Fully leverage the green and environmentally friendly characteristics and high efficiency and economic advantages of microbial remediation technology: This invention only requires one inoculation treatment during the seedling stage, without the need for additional chemical agents. In addition, combined with the characteristics of Penicillium oxalate SL2, this method also has multiple potentials such as reducing the amount of chemical phosphate fertilizer applied, maintaining the stability of soil physicochemical properties, and promoting nutrient cycling and energy flow. It has good ecological benefits and has good application prospects in the fields of phosphate-solubilizing bacterial fertilizer development, risk management and safe utilization of lead pollution in farmland soil. Attached Figure Description
[0021] Figure 1 This is a comparison chart of lead content in various tissues (roots, stems, leaves, husks, and grains) of rice at maturity in Comparative Example 1 and Example 1 of this invention. Figure 2 This is a comparison chart of lead translocation coefficients among different parts of rice in Comparative Example 1 and Example 1 of this invention. In the chart, a is the TF translocation coefficient from rice root to stem, b is the TF translocation coefficient from rice stem to leaf, c is the TF translocation coefficient from rice stem to husk, and d is the TF translocation coefficient from rice stem to grain. Figure 3 This is a comparison chart of the fresh weight of whole plant biomass (a) and fresh weight of single rice panicle (b) at maturity in Comparative Example 1 and Example 1 of this invention. Figure 4 The figures shown are comparative diagrams of lead distribution in the iron membrane and subcellular components of rice roots in Comparative Example 1 and Example 1 of this invention. Figure a shows the distribution of lead in the iron membrane, cell wall, organelles, and soluble components of rice roots, while Figure b shows a comparative diagram of lead distribution in the iron membrane, cell wall, organelles, and soluble components of rice roots. Detailed Implementation
[0022] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.
[0023] All other reagents in the following examples can be commercially available products of analytical grade or higher.
[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0025] The Penicillium oxalicum SL2 strain used in the following embodiments of the present invention is preserved in the China Center for Type Culture Collection (CCTCC), with the preservation number CCTCC NO: M 2014505, and the strain name: Penicillium oxalicum SL2, preservation date: October 22, 2014.
[0026] Example 1 As Figures 1-4 shown, this example provides a method for promoting rice growth and controlling lead accumulation in grains by using Penicillium oxalicum SL2, specifically as follows: (1) Collect the surface soil of the farmland (weakly acidic clay loam, 5.5 < pH ≤ 6.5), air-dry it and pass it through a 10-mesh sieve to obtain an uncontaminated soil sample; (2) Referring to the risk control values of the "Soil Environmental Quality - Risk Control Standards for Agricultural Land Soil Pollution" (GB / T 15618-2018), uniformly mix the Pb(NO3)2 solution into the soil (Pb content is 500 mg / kg), age and air-dry it for more than 12 months, and pass it through a 10-mesh sieve to obtain a lead-contaminated soil sample; (3) Inoculate Penicillium oxalicum SL2 into a potato dextrose agar medium (PDA) and culture it at 28 °C for 7 days, harvest fresh spores, and prepare a mother spore suspension with a 0.01% sterile Triton X-100 solution, and configure a spore suspension with a concentration of 1×10 6 spores / mL; (4) Select 14-day-old rice seedlings with consistent growth (a total of 18 plants), wash the roots and immerse them in the above spore suspension with a height of 5 cm, and co-culture them for 7 days under the conditions of 28 °C, 16 h light / 8 h darkness; (5) Transplant to the lead-contaminated soil sample synchronously with the control group, and use the water management mode of flooding before the heading stage (60 days, maintaining a field water layer of 2 - 3 cm) - alternating flooding and draining at the heading stage (irrigate shallow water of 2 - 3 cm, wait for the field water layer to naturally drain for 1 - 2 days and then re-water, cycle alternately) to cultivate the rice until maturity, and harvest the rice plants on the 100th day after transplantation; collect all the grains of each rice plant, weigh and record the spike weight per plant. Wash the rice plants with deionized water,吸干表面水分后测量并记录整株鲜重。将水稻根、茎、叶、籽粒等各部位分离并分别装入自封袋,保存于超低温冰箱(-80 °C)备用;(吸干表面水分后测量并记录整株鲜重。将水稻根、茎、叶、籽粒等各部位分离并分别装入自封袋,保存于超低温冰箱(-80 °C)备用;这部分中文原文表述不太准确清晰,翻译时尽量根据理解调整为合理内容)吸干 the surface moisture and measure and record the fresh weight of the whole plant. Separate each part of the rice plant, such as roots, stems, leaves, and grains, and put them into self-sealing bags respectively, and store them in a ultra-low temperature refrigerator (-80 °C) for standby; (6) After digesting each part of the rice, use an inductively coupled plasma mass spectrometer (ICP-MS) to measure the Pb content, and calculate the transfer coefficient (TF) between each part according to the measurement results. The transfer coefficient is the ratio of the Pb content in the above-ground part to the Pb content in the root part; (7) After weighing the root samples, the iron plaque on the root surface was extracted with DCB solution. The rice root samples after extracting the iron plaque were subjected to subcellular fraction extraction by differential centrifugation. After digestion, volume fixation, and filtration of each fraction, the Pb content was determined by ICP-MS, and the distribution ratio of the Pb content in each fraction in each subcellular fraction was calculated.
[0027] Comparative Example 1 As Figures 1-3 shown, this comparative example provides a rice plant not treated with the Penicillium oxalicum SL2 inoculant compared with the embodiment, specifically as follows: (1) The topsoil of the farmland (weakly acidic clay loam, 5.5 < pH ≤ 6.5) was collected, air-dried, and passed through a 10-mesh sieve to obtain an uncontaminated soil sample; (2) Referring to the risk control value of the "Soil Environmental Quality - Risk Control Standards for Agricultural Land Soil Pollution" (GB / T 15618-2018), Pb(NO3)2 solution was evenly mixed into the soil (Pb content was 500 mg / kg). After aging and air-drying for more than 12 months and passing through a 10-mesh sieve, a lead-contaminated soil sample was obtained; (3) Eighteen 14-day-old rice seedlings with consistent growth were selected. After washing the roots, they were immersed in Kimura nutrient solution without spores with a height of 5 cm and co-cultured for 7 days under the conditions of 28 °C, 16 h light / 8 h darkness; (4) Transplanted to the lead-contaminated soil sample synchronously with the embodiment, the rice was cultivated to maturity using a water management mode of flooding before the heading stage (60 days, maintaining a 2-3 cm water layer in the field) - alternating flooding and drying at the heading stage (irrigating shallow water of 2-3 cm, waiting for the water layer in the field to naturally dry for 1-2 days and then re-watering, cycling alternately). The rice plants were harvested on the 100th day after transplantation; all the grains of each rice plant were collected, weighed, and the single-plant panicle weight was recorded. The rice plants were washed with deionized water, the surface moisture was blotted dry, and the fresh weight of the whole plant was measured and recorded. Each part of the rice, such as roots, stems, leaves, and grains, was separated and put into self-sealing bags and stored in an ultra-low temperature refrigerator (-80 °C) for later use; (5) After digestion of each part of the rice, the Pb content was determined by ICP-MS, and the transfer coefficient (TF) between each part was calculated; (6) After weighing the root samples, the iron plaque on the root surface was extracted with DCB solution. The rice root samples after extracting the iron plaque were subjected to subcellular fraction extraction by differential centrifugation. After digestion, volume fixation, and filtration of each fraction, the Pb content was determined by ICP-MS, and the distribution ratio of the Pb content in each fraction in each subcellular fraction was calculated.
[0028] As Figure 1As shown, the cumulative lead content in various tissues of rice at maturity in the examples and comparative examples is as follows: Root lead content ranged from 1106.50 to 1493.37 mg / kg, stem from 14.44 to 22.03 mg / kg, leaf from 10.68 to 17.06 mg / kg, husk from 0.40 to 0.89 mg / kg, and grain from 0.16 to 0.71 mg / kg. The overall trend of lead accumulation among rice tissues was root > stem > leaf > husk > grain. Compared with the comparative example, the cumulative lead content in all tissues of the rice plants in the examples, except for the husk, was significantly reduced (P < 0.5). Specifically, the average lead content in roots, stems, leaves, husks, and grains decreased by 13.9%, 20.9%, 24.4%, 32.8%, and 68.4%, respectively. In the example, the lead content of rice grains was 0.174 ± 0.012 mg / kg, which meets the lead limit of 0.2 mg / kg set for cereals in my country's National Food Safety Standard for Maximum Levels of Contaminants in Food (GB2762-2022).
[0029] like Figure 2 As shown, the Pb translocation coefficients between different parts of rice at maturity in the examples and comparative examples are as follows: Pb has a weak ability to migrate from the roots to the stem, and the TF translocation coefficient is 0.0128 - 0.0148. Figure 2 a); Pb has a strong ability to migrate from stem to leaf, with TF values all greater than 0.64 ( Figure 2 b); while the translocation coefficients of Pb from the stem to the husk and grain were less than 0.05 and 0.04, respectively, indicating that Pb has a weak ability to migrate into the edible parts of rice. Figure 2 (c-2d). In addition, the examples significantly reduced the Pb translocation coefficient from roots to stems, by 8.2% compared to the control; however, the examples had no significant effect on the Pb translocation coefficient among different tissues in the aboveground parts of rice (p > 0.05), indicating that Penicillium oxalate SL2 mainly reduces lead accumulation in grains by blocking lead translocation from roots to aboveground parts.
[0030] like Figure 3 As shown, the whole plant biomass (fresh weight) and fresh weight per panicle of rice at maturity in the examples and comparative examples are as follows: Compared with the comparative example, the whole plant biomass and single panicle weight of rice in the examples increased by 14.2% and 15.0%, respectively, indicating that Penicillium oxalicum SL2 can still significantly promote rice growth under lead stress.
[0031] like Figure 4As shown, the distribution of Pb in the iron membrane and subcellular components of rice roots in the examples and comparative examples is as follows: In the comparative example, the concentration and distribution ratio of Pb on the iron membrane of the root surface were the lowest, only 6.52%, with more than 90% of Pb entering the biological tissues of the rice roots. The cell wall was the largest site of Pb accumulation, followed by soluble substances and organelles. Compared with the comparative example, the proportion of Pb in the iron membrane of rice roots in the examples increased to 11.27%, and the absolute content of Pb increased by 63.7%; the distribution ratio of lead in the root cell wall increased from 39.49% to 52.48%, while the lead content in soluble substances and organelles decreased significantly. This indicates that *Penicillium oxalicum* SL2 effectively prevents the transport of lead into the cells and aboveground parts by strengthening the dual fixation effect of the iron membrane and root cell wall.
[0032] Furthermore, for the culture conditions used in this study, inoculation of rice seedlings only needs to be performed during the rice seedling stage to ensure successful colonization of SL2 in the rice rhizosphere. This promotes rice growth and reduces lead accumulation, fully leveraging the green, environmentally friendly, and economically efficient characteristics of microbial technology. Mechanistic studies show that SL2 effectively controls the translocation of lead to the aboveground and edible parts by strengthening the fixation of lead by the root surface iron membrane and root cell walls. Combined with the characteristics of Penicillium oxalate SL2, this method also has multiple potential benefits, including reducing the application of chemical phosphate fertilizers, maintaining soil physicochemical stability, and promoting nutrient cycling and energy flow. It has good ecological benefits and promising application prospects in the development of phosphate-solubilizing bacterial fertilizers and the risk management and safe utilization of lead pollution in farmland soils.
[0033] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.
Claims
1. A method for promoting rice growth and inhibiting lead accumulation in grains using Penicillium oxalate SL2, characterized in that, The method includes the following steps: S1: Preparation of Penicillium oxalate SL2 spore suspension, wherein Penicillium oxalate SL2 is... Penicillium oxalicum SL2, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2014505; S2: Immerse the roots of 14-day-old rice seedlings in the spore suspension at a depth of 4-6 cm and culture them for 7 days at 28°C under 16h light / 8h darkness conditions to colonize Penicillium oxalate SL2 in the rice rhizosphere. S3: Transplant the inoculated rice seedlings into lead-contaminated soil and cultivate them to maturity using a phased water management model.
2. The method for promoting rice growth and inhibiting lead accumulation in grains using Penicillium oxalate SL2 according to claim 1, characterized in that, The specific method for preparing the Penicillium oxalate SL2 spore suspension described in step S1 is as follows: Penicillium oxalate SL2 was inoculated into potato dextrose agar medium and cultured at 28°C for 7 days. Fresh spores were harvested and a spore suspension stock solution was prepared with 0.01% sterile Triton X-100 solution. The spore suspension stock solution was then diluted to the working concentration using Kimura nutrient solution.
3. The method for promoting rice growth and inhibiting lead accumulation in grains using Penicillium oxalate SL2 according to claim 2, characterized in that, The working concentration of the spore suspension is 1 x 10 5 ~ 1 x 10 7 ~ 1 x 10 4. The method for promoting rice growth and inhibiting lead accumulation in grains using Penicillium oxalate SL2 according to claim 3, characterized in that, The working concentration of the spore suspension is 1×10⁻⁶. 6 per mL.
5. The method for promoting rice growth and inhibiting lead accumulation in grains using Penicillium oxalate SL2 according to claim 1, characterized in that, The lead-contaminated soil is a weakly acidic farmland soil with a pH value of 5.5 to 6.5 and a total lead content of ≤500 mg / kg.
6. The method for promoting rice growth and inhibiting lead accumulation in grains using Penicillium oxalate SL2 according to claim 1, characterized in that, The phased water management model is as follows: before the heading stage, the field is continuously flooded for 60 days to maintain a water layer of 2-3 cm. After the heading stage, the rice is cultivated to maturity by alternating flooding and drying. The rice plants are harvested 100 days after transplanting.
7. A method for promoting rice growth and inhibiting lead accumulation in grains using Penicillium oxalate SL2 according to claim 6, characterized in that, The specific method of the flooding-drying alternation treatment is as follows: irrigate with shallow water 2-3cm, wait for the water layer in the field to dry naturally for 1-2 days, and then re-irrigate, repeating the cycle until maturity.
8. The application of a Penicillium oxalate strain SL2 in promoting rice growth and inhibiting lead accumulation in grains, characterized in that... The Penicillium oxalate strain SL2 is Penicillium oxalicum SL2, with accession number CCTCC NO: M 2014505.
9. A microbial inoculant for promoting rice growth and inhibiting lead accumulation in grains, characterized in that, The microbial agent uses *Penicillium oxalate* SL2 as the active ingredient, and the *Penicillium oxalate* SL2 strain is... Penicillium oxalicum SL2, with accession number CCTCC NO: M 2014505.
10. The microbial agent according to claim 9, characterized in that, The microbial agent is a spore suspension, and the concentration of the spore suspension is 1×10⁻⁶. 5 ~1×10 7 per mL.