油茶根际功能菌株的组合优化及菌肥研究
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摘要
油茶(Camellia oleifera)是我国重要的木本油料树种。由于油茶生长周期长,必须维持土壤肥力才能达到持续、高产的目的。微生物肥料可以改良土壤理化性质,调节植物生长,增加作物产量,且对生态环境友好。推广和应用微生物肥料是推动我国油茶产业升级的重要措施。
本实验对油茶根际固氮菌、溶磷菌、解钾菌的生态分布进行了研究,筛选出高效菌株；对这些菌株进行组合、优化发酵条件,以及肥效及微生态效应进行了研究。研究结果如下：
(1)油茶根际功能菌株的生态分布及分离筛选。对油茶根际及非根际土壤中的固氮、溶磷及解钾菌的分布进行了研究,结果发现：解钾菌呈现明显的根际效应(R/S=9.4),溶磷菌次之(R/S=2.75),固氮菌的根际效应最小(R/S=1.75)；分离纯化油茶根际的固氮、溶磷及解钾菌,并进行活力测定,结果表明：36株固氮菌中,菌株N11固氮活性最强(39.09mg/kg),其次是菌株N19(37.19mg/kg);15株溶磷菌,P1菌株活性最强(9.56mg/kg);73株解钾细菌中,菌株K50的活性最强(121.71 mg/kg),菌株K56(106.54 mg/kg)次之。
(2)功能菌株的组合及优化。对固氮细菌N11和N19、溶磷细菌P1、解钾细菌K50和K56,进行平板划线及液体混合培养发现两两间无拮抗作用；将五株功能菌株进行组合并测定活性,结果表明：N11+P1+K50+K56是一组固氮、溶磷、解钾活力较高的菌群组合。在阿须贝氏培养基的基础上,用蔗糖8g、葡萄糖10g、硝酸铵0.25g、NaCl0.3g,代替碳、氮源及NaCl,以接种量6%,装液量50mL(250mL三角瓶),30℃,140r/min,初始pH7.5的发酵工艺,最适宜固氮活性的发挥；在解无机磷培养基的基础上,用蔗糖12g、硝酸铵0.5g、酵母粉0.4g、NaC10.2g,代替碳源、氮源及NaCl,以接种量4%,装液量50mL(250mL三角瓶),30°C,140r/min初始pH7.0的发酵工艺,最适宜溶磷活性的发挥；在解钾细菌培养基的基础上,用葡萄糖8g、甘露醇5g、硝酸铵1.5g,牛肉膏0.5g,代替碳、氮源,以接种量4%,装液量50mL (250mL三角瓶),35℃,160r/min,初始pH7.5的发酵工艺,最适宜解钾活性的发挥。
(3)功能菌复合菌肥研制及其肥效研究。组合菌群按照不同活性要求在最佳条件下发酵1：1：1混合后,按照1：10吸附比例吸附于灭菌150min的膨润土和草炭(1：1)混合载体,初步制成功能菌复合菌肥。功能菌复合菌肥和化学肥料的不同的配比对油茶生长的影响,结果表明：功能菌复合菌肥(40%)+化学肥料(60%)混合施肥对油茶生长影响明显,苗高增幅45.4%,地径增幅19.1%,新生根数增幅27.4%。功能菌复合菌肥使油茶苗根际微生物数量平均提高了49.73%,土壤有机质平均提高了10.53%,土壤速效氮平均提高了8.68%,土壤速效磷平均提高了13.29%,土壤速效钾含量提高了11.56%。
Camellia oleifera is an important woody oil plant in China. Because of the long growth periods of Camellia, it is necessary to maintain the soil fertility for the purpose of sustainable and high yield. The microbial fertilizer, which is friendly to the ecological environment, can improve soil physical and chemical properties, regulate plants growth and increase the yield. Promotion and application of microbial fertilizer is an important measure to upgrade Camellia oleifera production in China.
In this study, we analyzed the ecological distribution of camellia rhizosphere nitrogen-fixing bacteria, phosphate-solubilizing bacteria and potassium-releasing bacteria and screened high-efficiency strains; meanwhile combined and optimized fermentation condition of the selected ones, analyzed the fertilizer efficiency and micro-ecological effects of the bacterial manure as well. The results as follow:
(1) Ecological distribution and selection of the functional bacterial strain from camellia rhizosphere. We analyzed the distribution of nitrogen-fixing bacteria, phosphate-solubilizing bacteria and potassium-dissoluting bacteria from rhizosphere and non-rhizosphere of camellia, the results showed that the potassium-releasing bacteria (R/S=9.4)> phosphate-solubilizing bacteria (R/S=2.75)> nitrogen-fixing bacteria (R/S=1.75). we separated and screened the former three kind of strains from the rhizosphere of Camellia, the results of activity test showed that the strain N11 has the strongest activity(39.09 mg/kg) among the 36 nitrogen-fixing strains, and the strain N19 (37.19 mg/kg) was secondary in activity; the strain P1 has the strongest activity (9.56 mg/kg) among the 15 phosphate-solubilizing strains; the strain K50 with activity (121.71 mg/kg) was the strongest among the 73 potassium-dissoluting strains, and strain K56 comes second with the activity (106.54mg/kg).
(2) Combination and optimization of these functional strains. There are no antagonistic reactions among the functional strains by streak cultivation and liquid mixed culture. Combined the 5strains functional bacteria and test the activity, we found that N11+P1+K50+K56 was a set of flora combination with higher nitrogen fixation, phosphorus dissolving and potassium releasing activity. On the base of Ashby culture medium, we took the cane sugar 8g, glucose 10g, ammonium nitrates 0.25g, NaCl 0.3g to replace carbon, nitrogen source and NaCl and cultured with inoculum concentration 6%, medium volume 50mL (250mL flask), temperature 30℃,140 r/min and pH7.5 to obtain the stronger nitrogen fixation activity;on the base of Pikovaskaia's culture medium, we took the sugar 12g, ammonium nitrates 0.5g, yeast powder 0.4g, NaCl 0.2g to replace the carbon, nitrogen source and NaCl, and cultured with inoculum concentration 4%, medium volume 50mL (250mL flask), temperature 30℃,140 r/min and pH7.0 to obtain the stronger phosphorus dissolving activity; on the base of the potassium bacteria releasing medium, we took the glucose 8g, mannitols5g, ammonium nitrates 1.5g, beef extract 0.5g to replace the carbon and nitrogen source, and cultured with inoculum concentration 4%, medium volume 50mL (250mL flask), temperature 35℃,160 r/min, and pH7.5 to obtain the higher potassium releasing activity.
(3) Study on development and effects of compound bacterial manure. The method of how we develop compound bacterial fertilizer is to mix the fermentation broth (1:1:1), which was fermented under optimal fermentation condition of each functional strain, was adsorbed with 1:10 adsorption proportion by the carrier which was the mixture of the bentonite and peat that sterilized in 150mins. Different ratio of compound bacterial manure and chemical fertilizer affect the growth of Camellia differently, we fertilized the camellia with the proportion that bacterial manure 40% and chemical fertilizer 60%, then the results showed that:the height of seedling increased 45.4%, stem base raise up 19,1%, and the quality of new root increased 27.4%. By using compound bacterial manure, the increment of rhizosphere microorganisms was 49.73%; the soil organic matter increased 10.53%, the nitrogen availability in soil increased 8.68%, the phosphorus availability raised up 13.29% and the increment of potassium availability in soil was 11.56%.
本实验对油茶根际固氮菌、溶磷菌、解钾菌的生态分布进行了研究,筛选出高效菌株；对这些菌株进行组合、优化发酵条件,以及肥效及微生态效应进行了研究。研究结果如下：
(1)油茶根际功能菌株的生态分布及分离筛选。对油茶根际及非根际土壤中的固氮、溶磷及解钾菌的分布进行了研究,结果发现：解钾菌呈现明显的根际效应(R/S=9.4),溶磷菌次之(R/S=2.75),固氮菌的根际效应最小(R/S=1.75)；分离纯化油茶根际的固氮、溶磷及解钾菌,并进行活力测定,结果表明：36株固氮菌中,菌株N11固氮活性最强(39.09mg/kg),其次是菌株N19(37.19mg/kg);15株溶磷菌,P1菌株活性最强(9.56mg/kg);73株解钾细菌中,菌株K50的活性最强(121.71 mg/kg),菌株K56(106.54 mg/kg)次之。
(2)功能菌株的组合及优化。对固氮细菌N11和N19、溶磷细菌P1、解钾细菌K50和K56,进行平板划线及液体混合培养发现两两间无拮抗作用；将五株功能菌株进行组合并测定活性,结果表明：N11+P1+K50+K56是一组固氮、溶磷、解钾活力较高的菌群组合。在阿须贝氏培养基的基础上,用蔗糖8g、葡萄糖10g、硝酸铵0.25g、NaCl0.3g,代替碳、氮源及NaCl,以接种量6%,装液量50mL(250mL三角瓶),30℃,140r/min,初始pH7.5的发酵工艺,最适宜固氮活性的发挥；在解无机磷培养基的基础上,用蔗糖12g、硝酸铵0.5g、酵母粉0.4g、NaC10.2g,代替碳源、氮源及NaCl,以接种量4%,装液量50mL(250mL三角瓶),30°C,140r/min初始pH7.0的发酵工艺,最适宜溶磷活性的发挥；在解钾细菌培养基的基础上,用葡萄糖8g、甘露醇5g、硝酸铵1.5g,牛肉膏0.5g,代替碳、氮源,以接种量4%,装液量50mL (250mL三角瓶),35℃,160r/min,初始pH7.5的发酵工艺,最适宜解钾活性的发挥。
(3)功能菌复合菌肥研制及其肥效研究。组合菌群按照不同活性要求在最佳条件下发酵1：1：1混合后,按照1：10吸附比例吸附于灭菌150min的膨润土和草炭(1：1)混合载体,初步制成功能菌复合菌肥。功能菌复合菌肥和化学肥料的不同的配比对油茶生长的影响,结果表明：功能菌复合菌肥(40%)+化学肥料(60%)混合施肥对油茶生长影响明显,苗高增幅45.4%,地径增幅19.1%,新生根数增幅27.4%。功能菌复合菌肥使油茶苗根际微生物数量平均提高了49.73%,土壤有机质平均提高了10.53%,土壤速效氮平均提高了8.68%,土壤速效磷平均提高了13.29%,土壤速效钾含量提高了11.56%。
Camellia oleifera is an important woody oil plant in China. Because of the long growth periods of Camellia, it is necessary to maintain the soil fertility for the purpose of sustainable and high yield. The microbial fertilizer, which is friendly to the ecological environment, can improve soil physical and chemical properties, regulate plants growth and increase the yield. Promotion and application of microbial fertilizer is an important measure to upgrade Camellia oleifera production in China.
In this study, we analyzed the ecological distribution of camellia rhizosphere nitrogen-fixing bacteria, phosphate-solubilizing bacteria and potassium-releasing bacteria and screened high-efficiency strains; meanwhile combined and optimized fermentation condition of the selected ones, analyzed the fertilizer efficiency and micro-ecological effects of the bacterial manure as well. The results as follow:
(1) Ecological distribution and selection of the functional bacterial strain from camellia rhizosphere. We analyzed the distribution of nitrogen-fixing bacteria, phosphate-solubilizing bacteria and potassium-dissoluting bacteria from rhizosphere and non-rhizosphere of camellia, the results showed that the potassium-releasing bacteria (R/S=9.4)> phosphate-solubilizing bacteria (R/S=2.75)> nitrogen-fixing bacteria (R/S=1.75). we separated and screened the former three kind of strains from the rhizosphere of Camellia, the results of activity test showed that the strain N11 has the strongest activity(39.09 mg/kg) among the 36 nitrogen-fixing strains, and the strain N19 (37.19 mg/kg) was secondary in activity; the strain P1 has the strongest activity (9.56 mg/kg) among the 15 phosphate-solubilizing strains; the strain K50 with activity (121.71 mg/kg) was the strongest among the 73 potassium-dissoluting strains, and strain K56 comes second with the activity (106.54mg/kg).
(2) Combination and optimization of these functional strains. There are no antagonistic reactions among the functional strains by streak cultivation and liquid mixed culture. Combined the 5strains functional bacteria and test the activity, we found that N11+P1+K50+K56 was a set of flora combination with higher nitrogen fixation, phosphorus dissolving and potassium releasing activity. On the base of Ashby culture medium, we took the cane sugar 8g, glucose 10g, ammonium nitrates 0.25g, NaCl 0.3g to replace carbon, nitrogen source and NaCl and cultured with inoculum concentration 6%, medium volume 50mL (250mL flask), temperature 30℃,140 r/min and pH7.5 to obtain the stronger nitrogen fixation activity;on the base of Pikovaskaia's culture medium, we took the sugar 12g, ammonium nitrates 0.5g, yeast powder 0.4g, NaCl 0.2g to replace the carbon, nitrogen source and NaCl, and cultured with inoculum concentration 4%, medium volume 50mL (250mL flask), temperature 30℃,140 r/min and pH7.0 to obtain the stronger phosphorus dissolving activity; on the base of the potassium bacteria releasing medium, we took the glucose 8g, mannitols5g, ammonium nitrates 1.5g, beef extract 0.5g to replace the carbon and nitrogen source, and cultured with inoculum concentration 4%, medium volume 50mL (250mL flask), temperature 35℃,160 r/min, and pH7.5 to obtain the higher potassium releasing activity.
(3) Study on development and effects of compound bacterial manure. The method of how we develop compound bacterial fertilizer is to mix the fermentation broth (1:1:1), which was fermented under optimal fermentation condition of each functional strain, was adsorbed with 1:10 adsorption proportion by the carrier which was the mixture of the bentonite and peat that sterilized in 150mins. Different ratio of compound bacterial manure and chemical fertilizer affect the growth of Camellia differently, we fertilized the camellia with the proportion that bacterial manure 40% and chemical fertilizer 60%, then the results showed that:the height of seedling increased 45.4%, stem base raise up 19,1%, and the quality of new root increased 27.4%. By using compound bacterial manure, the increment of rhizosphere microorganisms was 49.73%; the soil organic matter increased 10.53%, the nitrogen availability in soil increased 8.68%, the phosphorus availability raised up 13.29% and the increment of potassium availability in soil was 11.56%.
引文
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