Selection and validation of reference genes for quantitative real-time polymerase chain reaction analyses of Serratia ureilytica DW2

Background:Serratia ureilytica DW2 is a highly efficient phosphate-solubilizing bacteria isolated from Codonopsis pilosula rhizosphere soil that can promote the growth of C.pilosula;nonetheless,until now,no validated reference genes from the genus Serratia have been reported that can be used for the normalization of quantitative real-time polymerase chain reaction(RT–qPCR)data.Methods:To screen stable reference genes of S.ureilytica DW2,the expression of its eight candidate reference genes(16S rRNA,ftsZ,ftsA,mreB,recA,slyD,thiC,and zipA)under different treatment conditions(pH,temperature,culture time,and salt content)was assayed by RT–qPCR.The expression stability of these genes was analyzed using different algorithms(geNorm,NormFinder,and BestKeeper).To verify the reliability of the data,the expression of the glucose dehydrogenase(gdh)gene under different soluble phosphate levels was quantified using the most stably expressed reference gene.Results:The results showed that the zipA and 16S rRNA genes were the most stable reference genes,and the least stable genes were thiC and recA.The expression of gdh was consistent with the phosphate solubilization ability on plates containing the National Botanical Research Institute phosphate growth medium.Conclusion:Therefore,this study provides a stable and reliable reference gene of Serratia for the accurate quantification of functional gene expression in future studies.

Effect of Lanthanum on Solubilization of Rock Phosphate in Liquid Culture by Aspergillus Niger and Penicillium Oxalicum

Rock phosphate (RP) is a low efficient P fertilizer when directly used in the soil. Phosphate-solubilizing microorganisms (PSMs) can solubilize RP in fermentation or soil condition. The effect of different concentration of lanthanum (La) on the solubilization of RP was investigated by two isolates of phosphate-solubilizing fungi (PSF) Aspergillus niger P39 and Penicillium oxalicum P66 in liquid culture. Experimental results show that relatively higher concentration of La in the culture solution inhibites fungal growth and delays RP solubilizing activity of two isolates. This inhibitory effect of La on RP solubilization varies with PSF (isolate P66 is more sensitive to La than P39 in this experiment). Comparing the pH value of culture media with soluble P content as affected by La application, only within individual isolate not different isolates the negatively significant relationship was observed.

Single-cell Raman and functional gene analyses reveal microbial P solubilization in agriculture waste-modified soils

Application of agricultural waste such as rapeseed meal(RM)is regarded as a sustainable way to improve soil phosphorus(P)availability by direct nutrient supply and stimulation of native phosphate‐solubilizing microorganisms(PSMs)in soils.However,exploration of the in situ microbial P solubilizing function in soils remains a challenge.Here,by applying both phenotype‐based single‐cell Raman with D_(2)O labeling(Raman‐D_(2)O)and genotype‐based high‐throughput chips targeting carbon,nitrogen and P(CNP)functional genes,the effect of RM application on microbial P solubilization in three typical farmland soils was investigated.The abundances of PSMs increased in two alkaline soils after RM application identified by single‐cell Raman D_(2)O.RM application reduced the diversity of bacterial communities and increased the abundance of a few bacteria with reported P solubilization function.Genotypic analysis indicated that RM addition generally increased the relative abundance of CNP functional genes.A correlation analysis of the abundance of active PSMs with the abundance of soil microbes or functional genes was carried out to decipher the linkage between the phenotype and genotype of PSMs.Myxococcota and C degradation genes were found to potentially contribute to the enhanced microbial P release following RM application.This work provides important new insights into the in situ function of soil PSMs.It will lead to better harnessing of agricultural waste to mobilize soil legacy P and mitigate the P crisis.