不同水肥管理对红花生长的影响及红花根际解磷菌的筛选和鉴定

Effects of different water and fertilizer management measures on the growth of safflower and screening and identification of phosphorus-solubilizing bacteria in safflower rhizosphere

摘要:

【目的】分析不同水肥条件对红花生物量、根际土壤磷素及微生物的影响，并从红花根际土壤样品中分离具有高效解磷能力的菌株，为红花科学水肥管理提供理论依据，并为红花的生长发育和根际微环境研究提供优良菌株。【方法】采用不同磷肥梯度处理红花，在红花的莲座期、伸长期、盛花期和种子成熟期检测植株生物量，同时测定植株根际土壤微生物、全磷和速效磷以及土壤磷酸酶活性，并进行差异性和相关性分析。采用抖土法和稀释涂布法分离筛选具有高效解磷能力的菌株。通过16S rRNA基因序列比较分析，对其进行鉴定。通过钼锑抗比色法测定菌株在不同培养基中的溶磷能力。利用灌根法和稀释涂布法接种优势菌株，分析菌株在红花根际定殖能力和促生能力。【结果】W3-P2的水肥处理有利于红花生物量的积累，速效磷含量和磷酸酶活性随施加磷肥浓度的增加呈先增大后减小趋势，水分对土壤全磷、速效磷和磷酸酶的影响与红花发育时期相关。细菌是红花根际土壤的优势菌群，在种子成熟期W4-P2处理组细菌数目最多，分别为3.017×10⁷ CFU/g和3.021×10⁷ CFU/g，远高于相同处理组的真菌和放线菌。从红花根际土壤筛选出5株高效解磷菌株（登录号C1：OR493125；C2：OR493126；C5：OR493127；C6：OR493128；C7：OR493129），均对以无机磷和有机磷为唯一磷源的培养基具有溶磷能力和降低pH的功能，其中C6的溶磷能力最强，在磷酸三钙、磷酸铝、磷酸铁和植酸钙无机磷培养基中解磷量分别为380.00、269.32、7.15、48.16 mg/L，在有机磷（卵磷脂）培养基中解磷量为18.19 mg/L。通过16S rRNA基因序列分析，C6为假单胞菌属，C1、C2、C5、C7为中华根瘤菌属。在红花植株周围接种2%优势解磷菌C1、C5和C6菌体悬液（10⁸ CFU/mL），在21 d时仍然保持在10⁵ CFU/g，其中C6定殖能力最强。同时检测盛花期生物量（叶片数、株高、茎粗、茎秆重和根长），结果显示均能显著促进红花生长，其中C6菌株促生能力最强，分别为122片、115.96 cm、12.49 mm、43.36 g、21.17 cm。【结论】水肥影响红花根际微环境的速效磷含量和微生物数目的变化水平，促进红花根系的生长发育，从而直接或间接影响红花生物量，W3-P2的水肥量相对适合红花的生长。菌株C6是一株高效解磷菌株，能够分解难溶性有机磷和无机磷，盆栽实验表明C6可以在红花根际定殖并显著促进红花生长。

Abstract:

[Objective] To study the effects of different water and fertilizer conditions on the biomass of safflower and the phosphorus and microorganisms in the rhizosphere soil, and isolate the strains with high phosphorus-solubilizing ability from the rhizosphere soil samples, so as to provide a theoretical basis for the reasonable water and fertilizer management of safflower and excellent strains for the research on safflower growth and rhizosphere microenvironment. [Methods] The safflower plants were treated with phosphorus fertilizer at different gradients, and the plant biomass was determined at the rosette, jointing, blooming, and seed maturity stages. At the same time, the microbial community composition, total phosphorus, available phosphorus, and phosphatase activity in the rhizosphere soil were determined. The strains with high phosphorus-solubilizing ability were screened by soil shaking and dilution coating methods and preliminarily identified by comparative analysis of 16S rRNA gene sequences. The phosphorus-solubilizing abilities of the strains in different media were determined by the molybdenum antimony colorimetric method. Furthermore, we inoculated the dominant strains by using the root irrigation method and dilution coating method to analyze the abilities of the strains to colonize the rhizosphere and promote the growth of safflower. [Results] W3-P2 water and fertilizer treatments were conducive to the biomass accumulation of safflower. As the application amount of phosphorus fertilizer increased, the available phosphorus content and phosphatase activity showed a trend of first increasing and then decreasing. The effects of water on soil total phosphorus, available phosphorus, and phosphatase activity were correlated with the safflower development stages. Bacteria were the dominant microorganisms in the rhizosphere soil of safflower, with the highest counts of 3.017×10⁷ CFU/g and 3.021×10⁷ CFU/g at the seed maturity stage in the water treatment and phosphorus fertilizer treatment groups, respectively, which were much higher than those of fungi and actinomycetes in the same treatment group. Five efficient phosphorus-solubilizing strains (accession number C1:OR493125; C2:OR493126; C5:OR493127; C6:OR493128; C7:OR493129) were screened out from the rhizosphere soil of safflower. All of them had the ability to solubilize phosphorus and lowered the pH of the medium with inorganic and organic phosphorus as the only phosphorus source. Strain C6 had the strongest ability to solubilize phosphorus, with the amounts of phosphorus solubilized in the inorganic phosphorus media of tricalcium phosphate, aluminum phosphate, ferric phosphate, and calcium phytate being 380.00, 269.32, 7.15, and 48.16 mg/L, respectively. It solubilized 18.19 mg/L phosphorus in the medium with organic phosphorus (lecithin) as the only phosphorus source. C6 was identified by 16S rRNA gene sequencing analysis as Pseudomonas sp. and C1, C2, C5, and C7 as Sinorhizobium sp. After inoculation of 2% suspensions (10⁸ CFU/mL) of the dominant phosphorus-solubilizing bacterial strains C1, C5, and C6 around safflower plants, the cell count remained 10⁵ CFU/g on day 21, and C6 demonstrated stronger colonization. The strains increased the leaf number, plant height, stem diameter, stem weight, and root length of safflower at the blooming stage, and the C6 strain group presented stronger effects, with the indicators above being 122 tablets, 115.96 cm, 12.49 mm, 43.36 g, and 21.17 cm, respectively. [Conclusion] Water and fertilizer management affects the available phosphorus content and microbial community structure in the rhizosphere microenvironment of safflower and promotes the growth and development of safflower roots, thus directly or indirectly affecting the biomass of safflower. The irrigation at 3 000 m³/hm² and phosphorus fertilizer application 375 kg/hm² were suitable for the growth of safflower. C6 is an efficient phosphorus-solubilizing strain, capable of decomposing insoluble organic and inorganic phosphorus. Pot experiments showed that C6 could colonize the rhizosphere of safflower and significantly promoted the growth of safflower.