长期有机无机配施下烤烟-玉米轮作优化土壤微生物活化无机磷

Optimization of Soil Microbial Activation of Inorganic Phosphorus in Tobacco-maize Rotation Under Long-term Combined Application of Organic and Inorganic Fertilizers

采集遵义市15年长期施肥定位试验的土壤,包括撂荒地(对照)、烤烟连作-单施化肥、烤烟连作-化肥有机肥配施、烤烟玉米轮作-单施化肥和烤烟玉米轮作-化肥有机肥配施等处理,通过土壤悬浊液与培养液混合培养技术、微生物高通量测序及常规分析,研究长期施肥对土壤无机磷活化的影响。结果表明,烤烟玉米轮作、施用有机肥和撂荒地的土壤可培养微生物(细菌、真菌、放线菌和无机磷细菌)数量增多,种群多样性增加,说明土壤生态环境改善。将土壤悬浊液接种至微生物培养液中,培养期间混合液中的有效磷含量以烤烟玉米轮作高于烤烟连作,化肥有机肥配施和撂荒地高于单施化肥,其中以烤烟玉米轮作-化肥有机肥配施最高,烤烟连作-单施化肥最低,说明合理种植和科学施肥有益于土壤无机磷的活化,在遵义烟区提倡烤烟玉米轮作-化肥有机肥配施很有必要。在混合液中,氢离子与溶解钙磷、有机酸与溶解铁磷和铝磷显著相关。与溶磷微生物的纯培养技术相比,土壤悬浊液培养既能反映微生物的溶磷特征,又更接近土壤微生物菌群复杂的溶磷状况。在供试土壤微生物中,存在柠檬酸合酶、蛋白葡萄糖脱氢酶和苹果酸合酶等的合成通路,处理间的差异可从分子生物学角度解释合理轮作和化肥有机肥配施促进土壤无机磷活化的原因。

【Objective】Mixed culture technology of soil suspension and culture medium,high-throughput sequencing,and conventional analysis methods were used to study the influence of long-term fertilization on inorganic phosphorus(P)mobilization in soil.【Method】Soil samples were collected from the plots of a 15-year-long experiment regarding crop cultivation and fertilization in Zunyi City,China.The treatments included(i)fallow soil(Control),(ii)consecutive flue-cured tobacco cultivation plus the application of only chemical fertilizers(C-CF),(iii)consecutive flue-cured tobacco cultivation plus the application of chemical fertilizers in combination with manures(C-CFM),(iv)flue-cured tobacco-maize rotation plus the application of only chemical fertilizers(R-CF),and(v)flue-cured tobacco-maize rotation plus the application of chemical fertilizers in combination with manures(R-CFM).The soil collected from the long-term fertilization test site was divided into two parts.A part of the soil sample was used for the mixed culture technology of soil suspension and culture solution.The method involved preparing the soil suspension(soil︰sterile water=1︰100)and then diluting it to 10^(-5) with sterile water.200μL diluent was taken and inoculated into NBRIP(National Biological Research Institute phosphate growth medium)solid medium with Ca_(3)(PO_(4))_(2),FePO_(4) and AlPO_(4) as phosphorus sources.After incubation at 30℃ for 2 days,the number of colonies with a phosphorus dissolving circle was observed.Then,another 200μL 1︰100 soil suspension was taken and inoculated in 50 mL NBRIP liquid medium with Ca_(3)(PO_(4))_(2),FePO_(4) and AlPO_(4) as phosphorus sources for 16 days.At 12,24,48,72,96,120 and 144 hours after inoculation,the culture medium was drawn,and the pH,soluble inorganic phosphorus content,and organic acid content were determined.The other part of soil samples was sent to Meggie Biological Company for high-throughput sequencing to study the effect of long-term fertilization on the relative abundance of enzyme synthesis pathway related to organic acid synthesis.【Result】The number of cultivable microorganisms(bacteria,fungi,actinomycetes and inorganic phosphate bacteria)in flue-cured tobacco-maize rotation,application of organic fertilizer and Control soil increased,and the population diversity increased.When the soil suspension was inoculated in a microbial culture medium,the content of available phosphorus was higher in the flue-cured tobacco-maize rotation than in consecutive flue-cured tobacco cultivation.Also,the available phosphorus was higher in the application of chemical fertilizers in combination with manures and Control than when only chemical fertilizers were applied;among which R-CFM was the highest and C-CF was the lowest.In the mixture,hydrogen ions were significantly correlated with dissolved Ca_(3)(PO_(4))_(2),and organic acids were significantly correlated with dissolved FePO_(4) and AlPO_(4).This indicates that compared with pure culture technology of phosphorus-mobilizing microbes,soil suspension culture can not only reflect the phosphorus-mobilizing characteristics of microorganisms but also be closer to the complex phosphorus solubilization status of soil microbial flora.In high-throughput sequencing,the relative abundance of the synthetic pathways of citrate(Si)-synthase,protein glucose dehydrogenase and malate synthase in the soil of R-CFM was significantly higher than that of other treatments.【Conclusion】Optimal crop cultivation and scientific fertilization are conducive to the activation of soil inorganic phosphorus,and R-CFM can be promoted as a flue-cured tobacco demonstration cultivation model for widespread promotion in Zunyi tobacco areas.Soil suspension culture can be used as an extension of pure microbial liquid culture as(a)it is closer to the complex phosphorus solubilization status of soil microbial flora,(b)effectively characterizes the ability of PMM to activate inorganic phosphorus,and(c)supplements the deficiencies of pure microbial liquid culture technology.The difference in the relative abundance of enzyme synthesis pathways related to the synthesis of organic acids also explains the reason for rational crop rotation and combined application of chemical fertilizers and manures to promote the activation of soil inorganic phosphorus from the perspective of molecular biology.