Vol. 63, 2017, No. 12: 574–580 Plant Soil Environ. doi: 10.17221/590/2017-PSE Which of soil microbes is in positive correlation to yields of maize (Zea mays L.)? Zhongyou MA1, Yue XIE1,2, Lin ZHU3, Liang CHENG1, Xin XIAO1, Cheng ZHOU1,4,*, Jianfei WANG1,* 1Key Laboratory of Bio-organic Fertilizer Creation, Ministry of Agriculture, Anhui Science and Technology University, Bengbu, P.R. China 2Department of Plant Pathology, North Carolina State University, Raleigh, USA 3School of Life Science and Technology, Tongji University, Shanghai, P.R. China 4Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, P.R. China *Corresponding authors: [email protected]; [email protected] ABSTRACT Ma Z.Y., Xie Y., Zhu L., Cheng L., Xiao X., Zhou C., Wang J.F. (2017): Which of soil microbes is in positive correla- tion to yields of maize (Zea mays L.)? Plant Soil Environ., 63: 574–580. Soil microorganisms are critical to maintain soil function, enhance plant health and increase crop yields. This study investigated the effects of organic matter on soil microbial community and assessed which of soil microbes were in positive correlation to maize yields. The results showed that different fertilizer treatments shaped specific microbial communities in the same soils. The most abundant beneficial soil microbes were found in treatments with organic fertilizer produced from cattle manure, return of wheat straw and 70% NPK admixture fertilizers treatment. The correlation analysis revealed that maize yields were in no correlation both to the shifts of soil microbial community structure and to the number of sequences or operational taxonomic units (OTUs) in soil microbes. However, maize yields were in positive correlation to microbial community structure shifts at the species level. 35 bacteria OTUs from 19 orders in 14 classes in 9 phyla were in positive correlation to yields of maize, while in fungi only one OTU25 belonging to Sordariales was in positive correlation. Our results indicate that the long-term application of organic and inorganic amendments could enrich the soil bacterial and fungal community and promote its diversity. Keywords: soil microbiome; plant-growth-promoting rhizobacteria; corn field; mineral fertilizer Soil microorganisms are critical to maintain soil Inoculation with plant-growth-promoting rhizo- function because of their contributions to soil bacteria (Azospirillum s-21) in the corn field in- structure formation, decomposition of organic creases dry weights of leaf, stem and grain (Gholami matter, toxin removal, and the biogeochemical et al. 2012). Organic inputs have a positive effect cycling of carbon, nitrogen, phosphorus and sul- on both bacterial and fungal diversity with or phur (Doran and Zeiss 2000, Zhao et al. 2014). without chemical fertilizers. Total diversity of Supported by the National Natural Science Foundation of China, Grant No. 31600210; by the Key Research Project of the Anhui Science and Technology Committee, Projects No. 15CZZ03102 and 1608085MC59; by the Research Founda- tion of Ministry of Agriculture, Grant No. BOFC2015KB02, and by the Research on the Application of Public Welfare Technology in Anhui Province, Projects No. 1604f0704045 and 1604f0704046. 574 Plant Soil Environ. Vol. 63, 2017, No. 12: 574–580 doi: 10.17221/590/2017-PSE bacterial and fungal communities is closely related total N (1.21 g/kg), total P (0.36 g/kg), and total to agro-ecosystem management practices and may K (115.48 mg/kg). Soil samples for isolating DNA partially explain the yield differences observed were collected at the summer maize harvest time between different treatments (Kamaa et al. 2011). in 2014. Many studies have been focused on exploring DNA extraction and PCR amplification. the effects of long-term fertilizer applications and Microbial DNA was extracted from soil sam- return of plant residues to agricultural fields on soil ples using the E.Z.N.A.® Soil DNA Kit (Omega physical properties (Pernes-Debuyser and Tessier Bio-tek, Norcross, USA) according to the man- 2004), soil fertility (Mallarino and Borges 2006), ufacturer’s protocols. The V4–V5 region of the soil organic matter and crop yields (Li et al. 2008, bacteria 16S rRNA gene was amplified by PCR Cao et al. 2011). Mineral fertilizer and organic (95°C for 2 min, followed by 25 cycles at 95°C manure addition can maintain or improve crop for 30 s, 55°C for 30 s, and 72°C for 30 s and a yields, and affect the soil microbial characteristics final extension at 72°C for 5 min) using primers under different fertilizer treatments. Structure of 515F 5'-barcode-GTGCCAGCMGCCGCGG)-3' maize (Zea mays L.) rhizosphere bacteria is evalu- and 907R 5'-CCGTCAATTCMTTTRAGTTT-3', ated to explore the feasibility of identifying novel where barcode is an eight-base sequence unique rhizosphere bacteria using culture-independent to each sample. The fungi 18S rRNA gene method based on direct amplification and analysis were amplified by PCR using primers 817F of 16S rRNA gene sequences and especially to ob- 5'-TAGCATGGAATAATRRAATAGGA-3' and tain a better understanding of bacterial community 1196R 5'-TCTGGACCTGGTGAGTTTCC-3'. structure and diversity from maize (Chauhan et Illumina MiSeq sequencing. Amplicons were al. 2011, Vega-Avila et al. 2015). extracted using the AxyPrep DNA Gel Extraction The functional soil microbiome can provide a Kit (Axygen Biosciences, Union City, USA) ac- sustainable and effective strategy to alter plant cording to the manufacturer’s instructions and morphology, enhance plant growth and increase quantified using QuantiFluor™-ST (Promega, crop yields. However, little is known about which Madison, USA). Purified amplicons were pooled in of soil microbes are related to yields of maize under equimolar and paired-end sequenced (2 × 250) on long-term fertilization and wheat-maize rotation an Illumina MiSeq platform. The raw reads were agro-ecosystem. The aim of this study was to inves- deposited into the NCBI Sequence Read Archive tigate the relationship between soil microbes and (SRA) database (Accession Number: SRR3099036- yields of maize by high-throughput sequencing of 3099047, SRR3099069, SRR3099089, SRR3099110, soil microbial metagenomic analyses, and to find SRR3099129, SRR3099146, and SRR3100852). out the key microbes that influence maize yields. Processing of sequencing data. Raw fastq files were demultiplexed, quality-filtered using QIIME version( 1.17, GitHub, San Francisco, USA). Operational MATERIAL AND METHODS taxonomic units (OTUs) were clustered with 97% similarity cutoff using UPARSE (http://drive5.com/ Experimental design. The field experiment uparse/) and chimeric sequences were identified and was established in 2011 to investigate the effects removed using UCHIME. The OTUs counts were of continuous application of farmyard manure, normalized into relative abundances (%). These crop residues, and chemical fertilizer (NPK) on software programmes were carried out with the soil microbial communities in a winter wheat- SPSS version 20.0 (IBM, Armonk, USA) for statisti- summer maize rotation. Micro plot trial for maize cal analysis, and R version 3.2.2 for drafts. Unless (Zea mays L.) was performed at the farm of the stated, P < 0.05 were considered significant. Linquan Agriculture Research Institute (32°55'N, 115°06'E), Anhui province, China as a randomized complete block design with 17 (Table 1) treatments RESULTS AND DISCUSSIONS replicated three times (three blocks). Each plot was a rectangle with an area of 50 m2 (5 × 10 m2). This study characterizes the soil microbial com- The averages of the soils characteristics were as munity composition of maize planted in rand- follows: pH 5.88, organic matter (12.64 g/kg), omized and replicated plots at harvest time in 575 Vol. 63, 2017, No. 12: 574–580 Plant Soil Environ. doi: 10.17221/590/2017-PSE Table 1. Experimental design of maize fertilization in different treatments Types and quantities of fertilizers (kg/ha) Fertilizer ingredients (kg/ha) Treatment NPK POF COF wheat straw BDA PIOF CIOF organic matter N P K W1 – – – – – – – 0 0 0 0 W2 307 – – – – – – 0 250.0 19.6 37.3 W3 215 3000 – – – – – 1362 244.0 42.1 56.0 W4 215 – 3000 – – – – 1524 244.3 32.5 86.4 W5 154 6000 – – – – – 2724 263.0 66.5 78.4 W6 154 – 6000 – – – – 3048 263.6 47.4 139.2 W7 307 – – 3000 – – – 2700 268.9 22.0 57.5 W8 307 – – 3000 150 – – 2835 269.8 22.1 58.5 W9 307 – – 3000 300 – – 2970 270.8 22.2 59.5 W10 215 3000 – 3000 – – – 4062 262.9 44.5 76.2 W11 215 – 3000 3000 – – – 4224 263.2 34.9 106.6 W12 154 6000 – 3000 – – – 5424 281.9 68.9 98.6 W13 154 – 6000 3000 – – – 5748 282.5 49.7 159.3 W14 – – – – – 1800 – 396 219.6 24.1 61.3 W15 – – – – – – 1800 450 217.6 22.4 59.4 W16 – – – – – 3600 – 792 439.2 48.1 122.6 W17 – – – – – – 3600 900 435.2 44.8 118.8 NPK – chemical fertilizer (250 kg/ha N + 20 kg/ha P + 37 kg/ha K); POF – kind of organic fertilizer produced from pig manure; COF – kind of organic fertilizer produced from cattle manure; BDA – biological decomposing agent, kind of microbial inoculum which can accelerate the decomposition and degradation of straw; PIOF – compound fertilizer was composed of chemical fertilizer and organic fertilizer produced from pig manure as the main raw material; CIOF – compound fertilizer was composed of chemical fertilizer and organic fertilizer produced from cattle manure as the main raw material; – indicates no addition 17 fertilizer treatments. The highest yield of maize gal diversity with or without chemical fertilizers was 10 037 kg/ha in W11, and the lowest yield (Kamaa et al. 2011). Organic fertilization regime was 3 672 kg/ha in W1. The variance of maize can maintain a stable tomato yield with reduced yields was significant in these different fertiliz- inputs of chemical fertilizers, which could be at- er treatments.