Analysis of microbial diversity in rhizosphere soil of Shine Muscat grape on different rootstocks using high-throughput sequencing

Abstract:【Objective】In order to provide reference for rootstock screening and cultivating, the community diversity of rhizosphere soil microorganisms of different rootstocks and self- rooted seedlings of Shine Muscat grape was explored. Rhizospheric microorganisms live at the interface between plant roots and soil and interact with plants. Beneficial rhizosphere microorganisms help plants to obtain nutrients and improve plant resistance to abiotic stresses. Harmful rhizosphere microorganisms compete with plants for nutrients in the soil or infect plants through roots, which further inhibits the healthy growth of plants. Plant roots and soil are two main factors affecting microbial community.【Methods】 Three-year-old Shine Muscat grape was used as the test material. There were three kinds of rootstock, including 1103P, 5BB and Beta, and self-rooted seedlings served as the control. Soil organic matter was determined by the potassium dichromate volumetric-dilution heat method. pH value was measured by 1∶1 water soil ratio and a pH meter. Alkaline hydrolysis of nitrogen was analyzed using the alkaline hydrolysis diffusion method. The available phosphorus was extracted by NaHCO3 and further determined by the molybdenum antimony sulfate anti colorimetry. Soil available potassium was extracted by ammonium acetate and then determined by the flame spectrophotometry. Exchangeable calcium and magnesium were extracted with 1 mol∙L^-1 NH4OAc solution, and the content of soluble calcium and magnesium was determined by the atomic absorption spectrophotometer. The total DNA of rhizosphere soil microorganisms was extracted using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.A.). The amplification primers of soil bacteria were 341F (5’-CCTAYGGGRBGCASCAG-3’) and 806R (5'-GGACTACNNGGGTATCTAAT-3’), and the amplification primers of soil fungi were ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS2R (5’-GCTGCGTTCTTCATCGATGC-3’). PCR reaction conditions were followed below: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, and extension at 72 ℃ for 30 s, 25 cycles; and extension at 72 ℃ for 5 min. The amplified products were recovered by AxyPrepDNA gel Recovery Kit (AXYGEN, U.S.A.). The purified PCR products were completely sequenced with high- throughput on Illumina PE250 platform (Shanghai Lingen Biotechnology Co., Ltd.). The Pair-end (PE) reads were spliced according to overlap relationship. Usearch software and gold database were used to remove the chimera by denovo and reference. After distinguishing samples, OTU clustering analysis and species taxonomy analysis were carried out. The RDP classifier Bayesian algorithm was used to classify 97% OTU representative sequences at similar levels (the confidence threshold was set at 0.7). The databases of Silva (for bacteria) and Unite (for fungi) were further employed, and the community composition of each sample was counted. 【Results】There was significant difference among the four samples (p＜0.05), in which the sample of 1103P had the lowest pH, while 5BB was the highest. The soil alkali-hydrolyzable nitrogen of 5BB and self-rooted seedlings was significantly higher than that of 1103P and Beta. The organic matter content of 1103P was lower than that of 5BB and self- rooted seedlings. High-throughput sequencing analysis showed that 1103P had the most abundant and diversified bacterial community in rhizosphere soil. Beta significantly increased the abundance and diversity of fungal community but reduced the diversity of bacterial community. The bacterial OTUs of the four samples were classified into 44 phyla, 133 classes, 345 orders, 536 families and 1056 genera. The dominant bacteria were Proteobacteria (24.15%-33.57% ), Acidobacteria (14.83%-22.82% ) and Actinobacteria (7.24%-10.99% ). Compared with selfrooted seedlings, rootstocks enhanced the relative abundance ratios of Acidobacteriota, Patescibacteria, and Verrucomicrobiota, but reduced the abundance ratios of Actinobacteriota and Gemmatimonadota, in which Acidobacteriota of 1103P and Beta increased by 53.87% and 40.54%, respectively. The dominant bacteria of 1103P were Candidatus Udaeobacter, RB41, and Nitrospira. The dominant bacteria of 5BB were Bryobacter, Chujaibacter and Sphingomonas. The dominant bacteria of Beta were Steroidobacter, Candidatus Udaeobacter and RB41. The dominant bacteria of self- rooted seedlings were Nitrospira, Bryobacter and Candidatus Udaeobacter. Compared with self- rooted seedlings, 1103P and 5BB had a totally different composition of bacterial community, while the composition of Beta and self-rooted bacterial community was similar. The fungal OTUs of four samples were classified into 8 phyla, 33 classes, 82 orders, 188 families and 412 genera. The dominant fungi were Ascomycota (55.57%-64.86%), Mucoromycota (5.84%-22.24%) and Basidiomycota (12.72%-19.22%), which were quite different among these four samples. The dominant fungi of 1103P were Mortierella, Fusarium, and Solicoccozyma. The dominant fungi of 5BB were Botryotrichum, Fusarium, and Tausonia. The dominant fungi of Beta were Fusarium, Mortierella, and Tausonia. The dominant fungi of self- rooted seedlings were Doratomyces, Mortierella, and Tausonia. The fungal OTU principal coordinates analysis indicated that there were significant differences in the composition of fungal communities among these four samples. The redundant analysis between bacterial community (phylum) and soil physical and chemical properties implied that the organic matter (OM) content, exchangeable magnesium (Mg2+ ) content, and pH value had a great impact on the dominant bacteria in rhizosphere soil.【Conclusion】Rootstocks changed the composition and quantity of rhizosphere soil microbial community of Shine Muscat, which favorably provides a theoretical basis for screening suitable rootstocks and improving the composition of rhizosphere microbial community.