大豆复合微生物肥料功能菌系的构建及包埋固定化研究
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摘要
化学肥料的应用虽然促进了作物产量的提高,但若长期大量施用,就会破坏土壤结构、导致土壤肥力下降,不但使增产效益明显下降,甚至会污染环境。和化肥相比,微生物肥料在提高肥料利用率、增加作物产量和保护生态环境方面具有明显的优势。深入开展微生物肥料研究,对于实现农业可持续发展具有非常重要的意义。为此,本文针对目前大豆接种剂活体菌功能单一和剂型局限性的问题,采取田问取样、室内分离鉴定、盆栽试验和化验分析相结合的手段,开展了大豆复合菌剂功能菌株筛选、菌系构建及包埋固定化研究,系统地评价了包埋菌剂的综合性能,以期为大豆新型微生物肥料品种的开发应用提供理论依据。
1.完成了田间采集样品的菌株分离、筛选、性能测试,并进行了分类鉴定：
(1)获得3株大豆根瘤菌R12、R6和R18,其中R12在促进根瘤数、根瘤干重和固氮酶活性增加,改善大豆生长性状,促进养分吸收和提高等方面,均优于参照菌USDA110; R12抗逆性优于USDA110,且表现出解磷活性；R6和R18不具有解磷活性,其他各项指标与参照菌USDA110相比或相等、或略低、或略高；3株菌都属于根瘤菌属(Rhizobium sp.), R12为菜豆根瘤菌(Rhizobium etli), R6和R18同为热带根瘤菌(Rhizobium tropici)的不同菌株。
(2)获得2株解磷细菌S7和S1。对测试的4种无机难溶磷酸盐溶P量S7为174.8mg/L、S1为167.3m∥L,较参照菌1203分别提高了5.87%和1.33%；对卵磷脂的解P量S7为49.33mg/L,S1为54.82mg/L,参照菌1203为52.93mg/L,解卵磷脂量S7略低于参照菌,S1略高于参照菌。相对而言,S7偏好溶无机磷,S1偏好解有机磷。S7和S1对无机和有机非溶性磷的总溶P量较参照菌分别提高了2.80%和1.88%。S7为芽孢杆菌属(Bacillus sp.)的成员,S1为假单胞菌属(Pseudomonas sp.)的成员。
(3)获得1株解钾菌株Cl,培养7d时其解K量为21.31mg/L,较参照菌L-K提高了34.28%；Cl为芽孢杆菌属(Bacillus sp.)的胶冻样芽孢杆菌(Bacillus mucilaginosus)。
2.对筛选获得的3类菌性能优良的菌株进行了拮抗性和生长关系试验,确定了能共处的菌株组合,优化了混合培养基质和培养条件,改进了培养方法：
(1)明确了R12、S7和Cl之间无生长抑制现象,3种菌混合培养时的生长关系为R12和S7、R12和Cl生长上相互促进,S7与Cl为无关共栖关系。
(2)优化后用于混合培养的基质成分为：甘露醇10g,酵母膏1.0g, NaCl0.1g,(NH4)2SO40.5g, K2HPO40.5g, KCl0.2g, MgSO4·7H2O1.0g, MnSO40.004g, CaCO35.0g, FeSO4·7H2O0.003g, CaCl20.1g,钾长石粉2.0g,磷矿粉5.0g,卵磷脂2.0g,Rh溶液4.0mL,蒸馏水1L。培养条件和培养方法为：按R12→S7→C1接种顺序,间隔12h,最适pH7.0,最适温度28℃。
3.进行了组合菌系包埋固定化试验,考察包埋材料组成对包埋操作、颗粒基本性能等指标的影响,并进行了不同剂型之间菌体抗逆性比较：
(1)初步确定包埋剂主料浓度及配比为SA3.0%-PVA3.0%,在此条件下,包埋的操作性、成球性,以及颗粒机械强度、传质性、包埋率、活菌数及其增殖倍数均较好。
(2)优化的包埋剂组成为SA3.0%-PVA(2.5%-3.0%)-(SiO24.0%-CaCO30.3%),在此条件下,操作性、成球性和传质性较好,所得包埋颗粒呈规则球形,直径为3mm-4mm,机械强度为53.4g/g-59.6g/g,包埋率为91.4%-94.3%,经过72h增殖培养,活菌数达到1011个/g,增殖倍数为500倍以上,活菌释放率达到90%以上。
(3)颗粒菌剂菌体的耐盐性、耐酸碱性、耐旱性、耐冷热性和耐药性等均较液体菌剂和草炭粉剂有明显的提高。
4.化肥、草炭粉剂、包埋菌剂的大豆盆栽比较试验,综合评价了菌剂效果,并明确了较佳的施用方法：
(1)菌剂的施用提高了大豆产量和品质,增加了大豆结瘤量、固氮量和养分吸收,改善了大豆生物学性状、土壤有效养分供应能力和土壤生物环境。
(2)菌剂与化肥配施互作效应显著,好于单施菌剂和单施同量化肥。
(3)包埋菌剂与半量化肥配施效果最好,各项指标均好于全量化肥处理；继续增加化肥配施量互作效应减弱。
(4)不论单施或与化肥配合施用包埋菌剂均好于草炭粉剂。
The application of chemical fertilizers could increase yield of crops, but it would destroy soil structure, emaciate soil fertility, ruduce production efficiency, even pollute the environment if large amount of chemical fertilizers was used for long time. Microbial fertilizers had obvious advantages in improving utilization ratio of fertilizer, increasing crop yield and protecting the ecological environment compared with chemical fertilizers. Hence, it has a great meaning for agricultural sustainable development to study microbial fertilizers deeply. Because of single function and dosage forms limitation with soybean, the objectives of this study were to screen functional strains, select non-antagonize strains into combination and then immobilize the microbial flora into particles, and evaluate the overall performances of the compound microbial fertilizer for soybean. This objectives have been achieved by the field sampling, lab analysis and pot experiment, and the results of this study might provide the theoretical basis for application and development of new-type microbial fertilizers for soybean.
1.Collected field samples, isolated and screened strains and tested their performances and identified:
(l) Three rhizobia strains with higher nodulation and nitrogenase activity were obtained, which were R12, R6and R18. R12was superior to the reference strain USDA110in many respects, for example, the number of nodules, nodule dry weight, nitrogenase activity, and growth promotion for plants. R12also exhibited organic phosphorus solubilization activity. Neither R6nor R18has phosphate solubilization activity. Other characters of R6and R18were approaching to USDA110. All of them were identified as the genus Rhizobium. Among them, R12identified as Rhizobium etli, and R6and R18as different strains of Rhizobium tropici.
(2)Two bacterial strains S7and S1with higher inorganic and organic phosphorus solubilization activity were obtained. The amout of dissolving insoluble phosphates of S7and S1was respectively174.8mg/L and167.3mg/L, which was5.87%and1.33%higher than that of reference strain1203. Decomposition amount of lecithin of S7and S1was respectively49.33mg/L and54.82mg/L, and which of the reference strain1203was52.93mg/L. In general, S7preferred to dissolving inorganic phosphorus, and S1preferred to decomposing organic phosphorus. The total amout of dissolved inorganic and organic phosphorus of S7and S1was2.80%and1.88%higher than that of the reference strain1203, respectively. S7was identified as the genus Bacillus, and S1as the genus Pseudomonas.
(3) One strain marked C1with high activity of decomposing soil potassium mineral were gotten. It could release potassium21.31mg/L into solution from soil potassium mineral when cultured seven days. The amount of decomposing soil potassium mineral of C1was34.28%higher than that of the reference strain L-K. C1was identified as Bacillus mucilaginosus of the genus Bacillus.
2.Selected the combination of strains in good performances by the antagonistic and growth relationship test for three types of strains, optimized the condition of culture and the medium component, and improved the method of culture.
(1)R12, S7and C1could coexist. R12-S7and R12-C1could promoting growth each other while mixed culture. However, S7-C1was in relationship of independent of habitat.
(2)Optimized medium formula was:mannitol10g, yeast extract1.0g,(NH4)2SO40.5g, K2HPO40.5g, KC10.2g, MgSO4·7H2O1.0g, MnSO40.004g, CaCO35.0g, FeSO4·7H2O0.003g, CaCl20.1g, potassium feldspar powder2.0g, rock phosphate5.0g, lecithin2.0g, Rh solution4.0mL, distilled water1L. The optimal order of inoculation was R12→S7→C1, the interval was12h. The optimal pH of mixed culture was7.0and culture temperature of mixed culture was28℃.
3.Immobilized the flora. Studied the influence of embedding material on embedding operation and basic characters of embedded particles, and compared the bacterial stress resistances in the different dosage forms:
(1)The basic ingredients composition was SA3.0%-PVA3.0%. Under the composition, operability and spheronization, as well as mechanical strength, mass transfer performance, embedded rate, the number of viable cells and their proliferation times were better than other ingredients composition.
(2) Under the optimized embedding medium [SA3.0%-PVA (2.5%-3.0%)-(SiO24.0%-CaCO30.3%)], operability, spheronization and mass transfer performance were clearly inproved. The particles presented regular ball with diameter of3-4mm, the mechanical strength was53.4-59.6g/g, and the embedded rate was91.4-94.3%. After72h proliferation, the number of viable cells reached magnitude1011per gram of the embedded particles. Proliferation multiples was500times more than no embedded strains, and the rate of releasing viable cells was90%higher than no embedded strains.
(3) The salt tolerance, the acid-base tolerance, the drought tolerance, the hot tolerance and the drug tolerance were stronger than no embedded strains.
4.Evaluated the effects of the microbial agents and ensured its application methods by the pot trial in which chemical fertilizer, peat inoculant and the embedded particles were respectively used.
(1) The yield and quality of soybean were improved, the amount of nodulations, nitrogen fixation and nutrient absorption were increased, and the biological characteristics of plants, available nutrient supplication and biological environment of soil were better by using the microbial agents than control treatment.
(2) The effects of applying the the microbial agents combined with chemical fertilizers were better than that of only using the same amount of chemical fertilizers or t the microbial agents.
(3) That the efficiency of combining the embedded particles and half amount of chemical fertilizers was better than that of full amount of chemical fertilizer treatment. But the interaction effects were weakened while increasing the amount of chemical fertilizers continuely.
(4) The effects of the embedded particles were always better than peat inoculant whether used alone or mixed with chemical fertilizers.
1.完成了田间采集样品的菌株分离、筛选、性能测试,并进行了分类鉴定：
(1)获得3株大豆根瘤菌R12、R6和R18,其中R12在促进根瘤数、根瘤干重和固氮酶活性增加,改善大豆生长性状,促进养分吸收和提高等方面,均优于参照菌USDA110; R12抗逆性优于USDA110,且表现出解磷活性；R6和R18不具有解磷活性,其他各项指标与参照菌USDA110相比或相等、或略低、或略高；3株菌都属于根瘤菌属(Rhizobium sp.), R12为菜豆根瘤菌(Rhizobium etli), R6和R18同为热带根瘤菌(Rhizobium tropici)的不同菌株。
(2)获得2株解磷细菌S7和S1。对测试的4种无机难溶磷酸盐溶P量S7为174.8mg/L、S1为167.3m∥L,较参照菌1203分别提高了5.87%和1.33%；对卵磷脂的解P量S7为49.33mg/L,S1为54.82mg/L,参照菌1203为52.93mg/L,解卵磷脂量S7略低于参照菌,S1略高于参照菌。相对而言,S7偏好溶无机磷,S1偏好解有机磷。S7和S1对无机和有机非溶性磷的总溶P量较参照菌分别提高了2.80%和1.88%。S7为芽孢杆菌属(Bacillus sp.)的成员,S1为假单胞菌属(Pseudomonas sp.)的成员。
(3)获得1株解钾菌株Cl,培养7d时其解K量为21.31mg/L,较参照菌L-K提高了34.28%；Cl为芽孢杆菌属(Bacillus sp.)的胶冻样芽孢杆菌(Bacillus mucilaginosus)。
2.对筛选获得的3类菌性能优良的菌株进行了拮抗性和生长关系试验,确定了能共处的菌株组合,优化了混合培养基质和培养条件,改进了培养方法：
(1)明确了R12、S7和Cl之间无生长抑制现象,3种菌混合培养时的生长关系为R12和S7、R12和Cl生长上相互促进,S7与Cl为无关共栖关系。
(2)优化后用于混合培养的基质成分为：甘露醇10g,酵母膏1.0g, NaCl0.1g,(NH4)2SO40.5g, K2HPO40.5g, KCl0.2g, MgSO4·7H2O1.0g, MnSO40.004g, CaCO35.0g, FeSO4·7H2O0.003g, CaCl20.1g,钾长石粉2.0g,磷矿粉5.0g,卵磷脂2.0g,Rh溶液4.0mL,蒸馏水1L。培养条件和培养方法为：按R12→S7→C1接种顺序,间隔12h,最适pH7.0,最适温度28℃。
3.进行了组合菌系包埋固定化试验,考察包埋材料组成对包埋操作、颗粒基本性能等指标的影响,并进行了不同剂型之间菌体抗逆性比较：
(1)初步确定包埋剂主料浓度及配比为SA3.0%-PVA3.0%,在此条件下,包埋的操作性、成球性,以及颗粒机械强度、传质性、包埋率、活菌数及其增殖倍数均较好。
(2)优化的包埋剂组成为SA3.0%-PVA(2.5%-3.0%)-(SiO24.0%-CaCO30.3%),在此条件下,操作性、成球性和传质性较好,所得包埋颗粒呈规则球形,直径为3mm-4mm,机械强度为53.4g/g-59.6g/g,包埋率为91.4%-94.3%,经过72h增殖培养,活菌数达到1011个/g,增殖倍数为500倍以上,活菌释放率达到90%以上。
(3)颗粒菌剂菌体的耐盐性、耐酸碱性、耐旱性、耐冷热性和耐药性等均较液体菌剂和草炭粉剂有明显的提高。
4.化肥、草炭粉剂、包埋菌剂的大豆盆栽比较试验,综合评价了菌剂效果,并明确了较佳的施用方法：
(1)菌剂的施用提高了大豆产量和品质,增加了大豆结瘤量、固氮量和养分吸收,改善了大豆生物学性状、土壤有效养分供应能力和土壤生物环境。
(2)菌剂与化肥配施互作效应显著,好于单施菌剂和单施同量化肥。
(3)包埋菌剂与半量化肥配施效果最好,各项指标均好于全量化肥处理；继续增加化肥配施量互作效应减弱。
(4)不论单施或与化肥配合施用包埋菌剂均好于草炭粉剂。
The application of chemical fertilizers could increase yield of crops, but it would destroy soil structure, emaciate soil fertility, ruduce production efficiency, even pollute the environment if large amount of chemical fertilizers was used for long time. Microbial fertilizers had obvious advantages in improving utilization ratio of fertilizer, increasing crop yield and protecting the ecological environment compared with chemical fertilizers. Hence, it has a great meaning for agricultural sustainable development to study microbial fertilizers deeply. Because of single function and dosage forms limitation with soybean, the objectives of this study were to screen functional strains, select non-antagonize strains into combination and then immobilize the microbial flora into particles, and evaluate the overall performances of the compound microbial fertilizer for soybean. This objectives have been achieved by the field sampling, lab analysis and pot experiment, and the results of this study might provide the theoretical basis for application and development of new-type microbial fertilizers for soybean.
1.Collected field samples, isolated and screened strains and tested their performances and identified:
(l) Three rhizobia strains with higher nodulation and nitrogenase activity were obtained, which were R12, R6and R18. R12was superior to the reference strain USDA110in many respects, for example, the number of nodules, nodule dry weight, nitrogenase activity, and growth promotion for plants. R12also exhibited organic phosphorus solubilization activity. Neither R6nor R18has phosphate solubilization activity. Other characters of R6and R18were approaching to USDA110. All of them were identified as the genus Rhizobium. Among them, R12identified as Rhizobium etli, and R6and R18as different strains of Rhizobium tropici.
(2)Two bacterial strains S7and S1with higher inorganic and organic phosphorus solubilization activity were obtained. The amout of dissolving insoluble phosphates of S7and S1was respectively174.8mg/L and167.3mg/L, which was5.87%and1.33%higher than that of reference strain1203. Decomposition amount of lecithin of S7and S1was respectively49.33mg/L and54.82mg/L, and which of the reference strain1203was52.93mg/L. In general, S7preferred to dissolving inorganic phosphorus, and S1preferred to decomposing organic phosphorus. The total amout of dissolved inorganic and organic phosphorus of S7and S1was2.80%and1.88%higher than that of the reference strain1203, respectively. S7was identified as the genus Bacillus, and S1as the genus Pseudomonas.
(3) One strain marked C1with high activity of decomposing soil potassium mineral were gotten. It could release potassium21.31mg/L into solution from soil potassium mineral when cultured seven days. The amount of decomposing soil potassium mineral of C1was34.28%higher than that of the reference strain L-K. C1was identified as Bacillus mucilaginosus of the genus Bacillus.
2.Selected the combination of strains in good performances by the antagonistic and growth relationship test for three types of strains, optimized the condition of culture and the medium component, and improved the method of culture.
(1)R12, S7and C1could coexist. R12-S7and R12-C1could promoting growth each other while mixed culture. However, S7-C1was in relationship of independent of habitat.
(2)Optimized medium formula was:mannitol10g, yeast extract1.0g,(NH4)2SO40.5g, K2HPO40.5g, KC10.2g, MgSO4·7H2O1.0g, MnSO40.004g, CaCO35.0g, FeSO4·7H2O0.003g, CaCl20.1g, potassium feldspar powder2.0g, rock phosphate5.0g, lecithin2.0g, Rh solution4.0mL, distilled water1L. The optimal order of inoculation was R12→S7→C1, the interval was12h. The optimal pH of mixed culture was7.0and culture temperature of mixed culture was28℃.
3.Immobilized the flora. Studied the influence of embedding material on embedding operation and basic characters of embedded particles, and compared the bacterial stress resistances in the different dosage forms:
(1)The basic ingredients composition was SA3.0%-PVA3.0%. Under the composition, operability and spheronization, as well as mechanical strength, mass transfer performance, embedded rate, the number of viable cells and their proliferation times were better than other ingredients composition.
(2) Under the optimized embedding medium [SA3.0%-PVA (2.5%-3.0%)-(SiO24.0%-CaCO30.3%)], operability, spheronization and mass transfer performance were clearly inproved. The particles presented regular ball with diameter of3-4mm, the mechanical strength was53.4-59.6g/g, and the embedded rate was91.4-94.3%. After72h proliferation, the number of viable cells reached magnitude1011per gram of the embedded particles. Proliferation multiples was500times more than no embedded strains, and the rate of releasing viable cells was90%higher than no embedded strains.
(3) The salt tolerance, the acid-base tolerance, the drought tolerance, the hot tolerance and the drug tolerance were stronger than no embedded strains.
4.Evaluated the effects of the microbial agents and ensured its application methods by the pot trial in which chemical fertilizer, peat inoculant and the embedded particles were respectively used.
(1) The yield and quality of soybean were improved, the amount of nodulations, nitrogen fixation and nutrient absorption were increased, and the biological characteristics of plants, available nutrient supplication and biological environment of soil were better by using the microbial agents than control treatment.
(2) The effects of applying the the microbial agents combined with chemical fertilizers were better than that of only using the same amount of chemical fertilizers or t the microbial agents.
(3) That the efficiency of combining the embedded particles and half amount of chemical fertilizers was better than that of full amount of chemical fertilizer treatment. But the interaction effects were weakened while increasing the amount of chemical fertilizers continuely.
(4) The effects of the embedded particles were always better than peat inoculant whether used alone or mixed with chemical fertilizers.
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