一株耐盐日本曲霉的筛选及其溶磷促生作用

Isolation,identification of a salt-tolerant,phosphate-solubilizing and crop-growth promoting Aspergillus japonicus

摘要:

[目的]从内蒙古种植葵花的盐碱地中筛选高效溶磷真菌，为盐碱地增产节肥开发生物肥料提供溶磷菌种资源。[方法]利用ITS rDNA序列鉴定菌株、固体培养基测定耐盐性，液体摇床培养与盆栽试验结合分析菌株溶磷能力，盆栽和田间试验明确菌株M1促进作物生长和增产作用；LC-MS技术测定菌株M1在液体培养基中分泌有机酸和植物激素含量，明确菌株M1的溶磷和促生机理。[结果]溶磷菌株M1鉴定为日本曲霉（Aspergillus japonicus）。液体培养基接种菌株M1培养6 d，以Ca₃（PO₄）₂为磷源时上清液有效磷达1020.89 mg/L，溶解率为63.30%；以AlPO₄为磷源时有效磷达995.69 mg/L，溶解率为48.59%；以贵州开阳磷矿粉、江苏锦屏磷矿粉、云南晋宁磷矿粉、河北钒山磷矿粉和云南昆阳磷矿粉为磷源接种菌株M1，从晋宁磷矿粉释放的有效磷浓度最高，达到363.64 mg/L。菌株M1可耐受10% NaCl。将M1制备的菌剂分别接种于施用Ca₃（PO₄）₂、AlPO₄和开阳磷矿粉3种磷源的4种盆栽试验土壤包括北京石灰性潮土、安徽黏性潮土、安徽水稻土和山东沿海盐潮土。结果显示，菌株M1对玉米植株促生效果显著，玉米植株鲜重比对照提高2.14%-90.91%、干重增加22.15%-268.28%；土壤有效磷提高21.81-24.27 mg/kg。菌株M1与4种土壤的适配性均高于对照菌株DSM 821。田间小区花生产量结果显示，接种溶磷菌剂M1增产效果最好，花生果实产量达4.46 t/hm²，比不接种菌剂的对照处理增加0.81 t/hm²，增产22.19%。菌株M1在含有磷酸三钙、磷酸铝和开阳磷矿粉3种难溶磷培养液中经过6 d培养，均产生7种有机酸，其中草酸和柠檬酸含量最高，分别为616.16 mg/L和413.69 mg/L；培养液均能检测到吲哚乙酸（IAA）和玉米素，IAA含量为15.45-77.58 mg/L，玉米素浓度为0.06-0.11 mg/L。[结论]获得了一株高效溶解多种难溶磷的日本曲霉菌M1，它能显著增加土壤有效磷、促进玉米生长和花生增产，与4种典型土壤适配性好，具有良好的农业应用前景。

Abstract:

[Objective] To isolate high effective phosphate-solubilizing microbial strains from growing sunflower in saline-alkali soils for bio-fertilizer production. [Methods] Phosphate-solubilizing fungus M1 was isolated by petri-dish methods and identified using ITS rDNA sequence homology analysis. The phosphate-solubilizing capacity of M1 was measured by broth medium and soil pot experiment. The effect of isolate M1 on plant growth promotion was studied in field experiment. Organic acids and phytohormone produced by isolate M1 were analyzed by means of LC-MS. [Results] Isolate M1 was identified as Aspergillus japonicus and had a strong ability to dissolve insoluble phosphates. The rate of Ca₃(PO₄)₂ dissolved by M1 was 63.30% and the concentration of available phosphorus was 1020.89 mg/L at 6 d shaking incubation. Soluble phosphorus in AlPO₄ liquid culture by M1was lower than in Ca₃(PO₄)₂ added medium, the content concentration of available phosphorus was 995.69 mg/L and the solubilized rate of AlPO₄was 48.59%.The greatest concentration of available phosphorus dissolved by isolate M1 from Jinning rock phosphate was 363.64 mg/L after 6 d shaking incubation. Isolate M1 combining salt resistance properties as well, the greatest content of NaCl was 10%. In greenhouse pot experiment, isolate M1 had a significant growth-promoting effect on corn in four kinds of soils (paddy soil, viscous fluvo-aquic soil, salinized fluvo-aquic soil and calcareous fluvo-aqui soil) under three kinds of insoluble phosphates treatment such as Ca₃(PO₄)₂, AlPO₄ and Kaiyang rock phosphate with 4 replicates per treatment. Compared with control (CK), inoculation with M1 increased the fresh weight of corn biomass by 2.14%-90.91% and dry weight of corn biomass by 22.15%-268.28%, and soil available phosphorus content increased 21.81-24.24 mg/kg. The adaptability of isolate M1 with four kinds of soils is greater than strain DSM 821. Field experiment showed that isolate M1 had a greater effect on enhancement of peanut yield, the yield was average 4.46 t/hm² and increased 22.19% being greater over control. 7 organic acids and 2 phytohormones were analyzed in liquid culture of Ca₃(PO₄)₂, AlPO₄ and Kaiyang rock phosphate. Out of seven acids, significant increases in the concentration of oxalic acid (616.16 mg/L) and citric acid (413.69 mg/L) were recorded in 3 liquid cultures by isolate M1, respectively. The concentration of indole acetic acid (IAA) was 15.45-77.58 mg/L, zeatin was 0.06-0.11 mg/L. [Conclusion] One new phosphate- solubilizing isolate M1 was obtained and identified as Aspergillus japonicus. Isolate M1 could solubilize insoluble phosphates in petri dishes, broth medium as well as pot experiments and increased soil available phosphate and corn biomass significantly in pot condition. So Aspergillus japonicus M1 strain have a better potential for bio-fertilizer production in the future.