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Study on the diversity of epiphytic bacteria on corn and alfalfa using Illumina MiSeq/NovaSeq high-throughput sequencing system

Study on the diversity of epiphytic bacteria on corn and alfalfa using Illumina MiSeq/NovaSeq... Purpose: To investigate the diversity of the epiphytic bacteria on corn (Zea mays) and alfalfa (Medicago sativa) collected in Hengshui City and Xingtai City, Hebei Province, China, and explore crops suitable for natural silage. Methods: The Illumina MiSeq/NovaSeq high-throughput sequencing system was used to conduct paired-end sequencing of the community DNA fragments from the surface of corn and alfalfa collected in Hengshui and Xingtai. QIIME2 and R software were used to sort and calculate the number of sequences and taxonomic units for each sample. Thereafter, the alpha and beta diversity indices at of species level were calculated, and the abundance and distribution of taxa were analyzed and compared between samples. Result: At phylum level, the dominant groups were Proteobacteria (70%), Firmicutes (13%), Actinobacteria (9%), and Bacteroidetes (7%). Meanwhile, the dominant genera were Pseudomonas (8%), Acinetobacter (4%), Chryseobacterium (3%), and Hymenobacter (1%). Enterobacteriaceae (24%) were the most predominant bacteria in both the corn and alfalfa samples. Alpha diversity analysis and beta diversity indices revealed that the diversity of epiphytic microbial communities was significantly affected by plant species but not by region. The diversity and richness of the epiphytic bacterial community of alfalfa were significantly higher than those of corn. Conclusion: This study contributes to the expanding knowledge on the diversity of epiphytic bacteria in corn and alfalfa silage and provides a basis for the selection of raw materials. Keywords: Bacterial diversity, Alfalfa, Corn Introduction facilitates animal digestion and absorption, increases the Natural silage is the process of converting the fermenta- value of forage utilization, expands the source of forage, tion substrate (soluble sugar) in raw materials into acidic and adjusts the forage supply period (Shang et al. 2019). products, such as lactic acid through the proliferation of After silage, the nutrients will not be reduced. Silage also lactic acid bacteria (LAB) on crops. This process creates has an aromatic and sour taste, which stimulates the ap- an acidic environment and inhibits the proliferation of petite of livestock and increases feed intake. The quality harmful microorganisms, thereby preserving the nutri- of natural silage was greatly affected by the epiphytic tional content of raw materials (Zhang et al. 2011). As a microflora on crops. The fermentation time of natural storage technology, silage reduces forage nutrient loss, silage is very long. LAB cannot form dominant bacteria in a short time. Therefore, the crops with high LAB and * Correspondence: liuguixia1971@163.com; tangshude1234@163.com low epiphytic microbial diversity should be more suit- School of Life Sciences, Hebei University, Baoding 071002, People’s Republic able for natural silage. of China Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Wu et al. Annals of Microbiology (2021) 71:38 Page 2 of 11 Studies have observed that LAB, such as Lactobacillus, content was approximately 50 ~ 60%. The specific sam- Lactococcus, Leuconstoc, Streptococcus, Pediococcus, and pling results are presented in Table 1. Enterococcus, play a key role during the silage fermenta- tion processing (Gharechahi et al. 2017). The abundance Sample DNA extraction and PCR amplification, of LAB on crops determines the success of the silage quantification, pooling and sequencing process. Depending on the production of metabolites, DNA was extracted using the E.Z.N.A.® Soil DNA Kit LAB can be divided into two groups: homofermentative (Omega Bio-tek, Norcross, GA, USA) according to LAB and heterofermentative LAB. Homofermentative the manufacturer’s protocols, and quantified using LAB can produce more lactic acid, which can improve Nanodrop. The quality of DNA extracted was ob- the fermentation quality of silage. Moreover, they pro- served through electrophoresis on a 1.2% agarose gel. duce volatile fatty acids to inhibit the growth of aerobic The variable region of the 16S rRNA gene (single or bacteria and improve the aerobic stability of silage. consecutive multiple) or specific gene fragments was The epiphytic microflora greatly affects the quality of amplified using polymerase chain reaction (PCR). natural silage fermentation. And the fermentation qual- Subsequently, the PCR products were purified using ity of silage can be affected by different crops or even Vazyme VAHTSTM DNA Clean Beads and quantified the same crop grown under different environments con- fluorometrically. Sequencing libraries were prepared ditions (Ali et al. 2020; Huang et al. 2019). Choosing the using Illumina TruSeq Nano DNA LT Library Prep appropriate forage ingredients can help improve the Kit. PCR products that met the minimum concentra- quality of the silage. Corn (Zea mays) and alfalfa (Medi- tion required for analysis were electrophoresed in 2% cago sativa) are often utilized to produce silage. Identify- agarose gel to check for the correct size of the target ing the epiphytic microflora on forage can provide a bands. High-throughput sequencing was conducted scientific basis for effectively regulating the fermentation using Illumina Miseq/NovaSeq at Personalbio Com- process of silage. The population of eubacteria on corn pany (Shanghai, China). samples collected immediately was mainly composed of genera belonging to Proteobacteria (56.4 ± 1.5%), specif- Bioinformatics and statistical analysis ically to orders Pseudomonadales, Xanthomonadales, Bioinformatics was mainly performed using QIIME2 and Enterobacteriales; and Bacteriodetes (37.4 ± 1.7%), (2019.4) (Bolyen et al. 2018). Sequences were denoised specifically to orders Sphingobacteriales and Flavobac- using the DADA2 plugin (Callahan et al. 2016). Instead teriales (Drouin et al. 2019). Lactobacillales were of clustering by similarity, DADA2 only performs dere- substantial contributors of Firmicutes, with Leuconosto- plication or clustering at 100% similarity. Because caceae representing between 60% and 100% of the fresh DADA2 has not yet been adapted to all amplicons, we forage sample composition (Drouin et al. 2019). Entero- retained the OTU clustering-based Vsearch (Rognes bacteriaceae are predominant on corn and alfalfa (Lin et al. 2016) as an alternative. The abundance, distribu- et al. 1992). Yeasts and moulds are also major epi- tion, alpha, and beta diversity indices were analyzed phytic microorganisms on both crops (Lin et al. 1992; using QIIME2 and R software (Xie et al. 2016). The ac- Zhang 2011). However, the abundance of LAB on the cession numbers for sequencing data presented is raw material for ensiling is far less than that of aerobic PRJNA745034. bacteria, Escherichia coli, yeast, mould, and other harmful microorganisms (Zhang et al. 2011). The number of Results LAB on the surface of corn was greater than that of Species composition analysis other raw materials (Cai et al. 1999; Kasmaei et al. The specific composition of the microbial communities 2017). However, there are few reports on the epiphytic at each classification level in each sample was obtained microorganisms on corn and alfalfa. This study discussed using the statistics of the amplicon sequence variant the species and diversity of epiphytic bacteria on corn (ASV) table after rarefaction. The ggplot2 package in R and alfalfa. (Ginestet 2011) was used to plot the data and generate a histogram to visualize the number of taxa at each classi- fication level in the samples (Fig. 1). A microbial classifi- Materials and methods cation hierarchy tree was also generated to reveal the Collection of samples composition of all taxa at the same time using R lan- Samples were collected in the budding to the early flow- guage ggtree package (Yu et al. 2017). ering stage of alfalfa, and in the late milking to the early At phylum level, Proteobacteria (70%), Firmicutes waxing stage of corn. Alfalfa and corn samples were col- (13%), Actinobacteria (9%), and Bacteroidetes (7%) were lected in Xingtai (113°52′ E, 36°50′ N) and Hengshui the most dominant (Fig. 2). The four phyla have more (115°10′ E, 37°04′ N), Hebei Province. The moisture branches in the tree, which indicates that the genotypes Wu et al. Annals of Microbiology (2021) 71:38 Page 3 of 11 Table 1 Origin and grouping of microbial samples Places Plants Breed Serial number Groups Xingtai Corn Jinchu 100 D1 D1-1 D1-2 D1-3 Duobao No. 3 D2 D2-1 D2-2 D2-3 Shengrui 565 D3 D3-1 D3-2 D3-3 Yawangqingchu No. 8 D4 D4-1 D4-2 D4-3 Alfalfa SR4030 E1 E1-1 E1-2 E1-3 Saidi 5 E2 E2-1 E2-2 E2-3 Zhongmu No. 1 E3 E3-1 E3-2 E3-3 Hengshui Corn Duobao No. 3 F1 F1-1 F1-2 F1-3 Shengrui 565 F2 F2-1 F2-2 F2-3 Jinchu 100 F3 F3-1 F3-2 F3-3 Yawangqingchu No. 8 F5 F5-1 F5-2 F5-3 Alfalfa SR4030 G1 G1-1 G1-2 G1-3 Zhongmu No. 1 G2 G2-1 G2-2 G2-3 Saidi 5 G5 G5-1 G5-2 G5-3 of the dominant bacteria in the samples are diversified Microbial diversity analysis of corn and alfalfa samples in evolutionary relations. Interestingly, Lactobacillales Alpha diversity analysis was more abundant in corn samples than in alfalfa sam- Alpha diversity represents the diversity of species within ples (Fig. 2). Enterobacteriaceae (24%), which belongs to a habitat. Chao1 species index measures the species rich- Proteobacteria, was the most predominant family in both ness within a community (Chao 1984), while Shannon corn and alfalfa samples. Four genera have more than and Simpson indices measure the species diversity 1% of the reads and they are Pseudomonas (8%), Acineto- within a community (Simpson 1949). Observed species bacter (4%), Chryseobacterium (3%), and Hymenobacter indices measure community richness. Phylogenetic di- (1%) (Figs. 2 and 3). versity (PD) index represents diversity based on Fig. 1 The number of taxa at each classification level for different samples Wu et al. Annals of Microbiology (2021) 71:38 Page 4 of 11 Fig. 2 The pie chart (threshold 0.5%) of each branch node in the classification tree shows the proportion of the taxon in each group. The larger the sector area, the higher the abundance of the taxon in the group. The percentage above the taxon represents the percentage of the total bacteria evolution (Faith 1992). Meanwhile, Pielou’s evenness 1958). Results were plotted into a boxplot using the R to index represents species evenness (Pielou 1996) and exhibit the differences in diversity indices observed be- Good’s coverage index represents the coverage (Good tween different groups (Fig. 4). The microbial Wu et al. Annals of Microbiology (2021) 71:38 Page 5 of 11 Fig. 3 Histogram of genus level species composition community richness, diversity, evenness, and evolution- were dispersed. Samples of corn (Fig. 5D, F) were similar ary diversity of alfalfa (Fig. 4E, G) were higher than that and the samples of alfalfa (Fig. 5E, G) were similar. The of corn (Fig. 4D, F). However, the coverage of species in results showed plant species affected the epiphytic bac- the microbial community of alfalfa was lower than that terial communities, as compared with region. of corn. The microflora of alfalfa and corn collected in Xingtai had significant differences in community rich- Species difference analysis and biomarker ness and evolutionary diversity. Meanwhile, the micro- The number of ASV in groups D, E, F, and G was 6683, flora of alfalfa and corn collected in Hengshui had highly 8305, 6920, and 8080 respectively (Fig. 6). There are 545 significant differences in community richness, diversity, ASVs in common, accounting for 2.34% of the total and evenness. Thus, the diversity of epiphytic bacterial ASV. community is significantly affected by plant species. All We utilized the relative abundances of the top 50 gen- alpha diversity indices in the microflora of alfalfa col- era to generate a heat map by heatmap package in R lected in both sites were not significantly different. (Zhao et al. 2014). Heatmap shows a data matrix where Alpha diversity indices, excluding evolutionary diversity, coloring gives an overview of the numeric differences. In in the microflora of corn collected in Xingtai and Heng- the genus-level species composition heat map for species shui were not significantly different. We can conclude clustering, red and blue patches indicate that the genera that the region has no significant effect on the diversity are more abundant and less abundant in a sample than of epiphytic microbial communities. the other sample. Lactic acid bacteria, such as those be- longing in the genera Leuconostoc and Lactobacillus, Beta diversity analysis have an important effect on silage fermentation and are The microbial communities in alfalfa and corn samples among the top 50 genera in terms of relative abundance. were compared using non-metric multidimensional scal- Leuconostoc was mainly present in groups D2, F1, F2, ing (NMDS) based on the weighted UniFrac distance and F3. Lactobacillus was mainly present in groups D1, (Lozupone and Knight 2005). Each point in the diagram D3, D4, F1, F2, F3, E1, E2, and E3 (Fig. 7). As presented represents a sample, and the different colored dots indi- in Fig. 7, groups F1 and F2 had higher abundance of cated different samples (Fig. 5). Samples were clustered Clostridium sensu stricto 1, which is harmful to according to their similarity, and the closer the distance fermentation. between two points is, the more similar the two samples We obtained the distribution of important species in are. Alfalfa group samples aggregated in the NMDS ana- each group by using the algorithm analysis of random lysis diagram, while the samples from the corn group forests (Breiman 2001) (Fig. 8). The abscissa represented Wu et al. Annals of Microbiology (2021) 71:38 Page 6 of 11 Fig. 4 Grouping box plot of alpha diversity index. The two groups below the two ends of the horizontal line indicate that the index of two groups are significant different. The number under the diversity index label is the P value of the Kruskal-Wallis test the importance of species to the classifier model, and Spirosoma, Hymenobacter, Bacillus, Actinomycetospora, the ordinate represented the taxon name at genus level. Taibaiella, and Sphingobacterium, can be considered The importance of genus in shaping the bacterial com- markers of differences in these groups. Most of these munity in each group decreases successively. These genera belong to Proteobacteria and Bacteroidetes. How- highly important genera, namely Pedobacter, Nocar- ever, LAB, which have a positive impact on fermentation dioides, Chryseobacterium, Burkholderia−Caballeronia were not observed. The presence of Bacillus is worth −Paraburkholderia, Paracoccus, Pseudomonas, Acineto- noting. Bacillus causes the silage to deteriorate, leading bacter, Allorhizobium−Neorhizobium−Pararhizobium to a rotten and smelly product. Bacillus was mainly ob- −Rhizobium, Larkinella, Mucilaginibacter, Sphingomo- served SR4030 and Saidi 5 in Xingtai. We should choose nas, Brevundimonas, Siphonobacter, Methylobacterium, crops with more LAB, less Bacillus and Clostridium, and Wu et al. Annals of Microbiology (2021) 71:38 Page 7 of 11 Fig. 5 NMDS analysis based on the weighted UniFrac distance lower epiphytic microbial diversity for natural silage, be- Drouin et al. (2019) observed that the populations of cause LAB on these crops may form dominant bacteria bacteria on the corn samples collected immediately after in a short time, reducing the loss of nutrients. Taking inoculation, but prior to ensiling, were mainly composed these factors into consideration, the epiphytic bacteria of genera belonging to Proteobacteria (56.4 ± 1.5%), spe- on Duobao No. 3 in Xingtai and Jinchu 100 in Hengshui cifically in orders Pseudomonadales, Xanthomonadales, may be better for natural silage. However, the content of and Enterobacteriales; and to Bacteriodetes (37.4 ± moisture, protein, and sugar of plants also influence the 1.7%), specifically in orders Sphingobacteriales and Fla- quality of silage. We are going to do further study to vobacteriales. In another study, 89.6% of the bacterial choose the crops that are suitable for natural silage. 16S rRNA gene sequences were associated with the phylum Proteobacteria, and 8.1% were associated with Discussion the phylum Firmicutes; other phyla identified were Acti- Corn and alfalfa are widely used raw materials for nat- nobacteria (0.2%) and Bacteroidetes (0.2%), before alfalfa ural silage. The epiphytic bacteria on crops substantially ensiling (McGarvey et al. 2013). The populations of bac- affect the quality of natural silage. Identifying the epi- teria in the fresh corn and alfalfa samples in previous phytic microflora on forage can provide a scientific basis studies were mainly composed of genera belonging to for effectively regulating the fermentation process of nat- Proteobacteria, Firmicutes, Actinobacteria, and Bacteroi- ural silage. There are only few detailed reports on the detes, although their abundance varied slightly (Ali et al. epiphytic microorganisms of corn and alfalfa. Most stud- 2020; Drouin et al. 2019; McGarvey et al. 2013). Most of ies reported the microbes using the plate count method the epiphytic bacteria of corn and alfalfa belong to Pro- (Cai et al. 1999; Lin et al. 1992). Drouin et al. (2019) teobacteria, and Enterobacteriaceae are the most pre- studied the epiphytic microflora on corn using high- dominant bacterial family on corn and alfalfa (Lin et al. throughput sequencing. However, these studies are not 1992). These results are consistent with the present sufficiently detailed. In this study, the Illumina MiSeq/ study. Proteobacteria was the most prevalent phylum in NovaSeq system was used to analyze the diversity of epi- fresh corn, and the bacterial community of alfalfa was phytic bacteria on corn and alfalfa. highly dominated by Firmicutes during the ensiling Wu et al. Annals of Microbiology (2021) 71:38 Page 8 of 11 Fig. 6 Venn diagram of sample (group) ASV period, when the aerobic environment was changed to 0.27, in the previous study (Drouin et al. 2019). We ob- anaerobic. Moreover, the abundance of Firmicutes in- served LAB, such as Leuconostoc and Lactobacillus,in creased significantly (Drouin et al. 2019). Sequences af- the top 50 genera in average abundance. Several studies filiated with Lactobacillales were substantial contributors have shown that corn has higher LAB composition than to the Firmicutes phylum order (Drouin et al. 2019). In other crops. For example, the number of LAB on the our study, the main epiphytic bacterial composition in surface of corn was twice than that of sorghum and al- corn and alfalfa were consistent with the results of previ- falfa, and 20 times than that of ryegrass (Cai et al. 1999). ous studies. Meanwhile, the total number of LAB on corn is seven Our results suggest that the diversity of epiphytic bac- times than that of grass and 15 times than that of clover terial communities is not affected by region, but it is sig- (Kasmaei et al. 2017). The lactic acid and acetic acid nificantly affected by plant species. This may also be contents of silage corn silage corn, elephant grass, and associated with the similar environments of the two sugarcane tops were significantly increased by adding sites; thus, environment cannot affect the epiphytic mi- the epiphytic microorganisms of corn straw, and the aer- croorganisms of the plants. However, the epiphytic mi- obic stability of elephant grass silage was positively af- croorganisms of forage are affected by forage species, fected (Huang et al. 2020). Kasmaei et al. (2017) stage of maturity, weather, mowing, field-wilting, chop- observed that the increase in lactic acid and acetic acid ping process humidity, solar radiation, plant surface content in corn straw silage by epiphytic microorgan- structure, and plant nutrient distribution (Lin et al. isms may be related to the abundance of Lactococcus 1992; Bai 2011). This may be the reason the species and and Leuconostoc (Kasmaei et al. 2017). Crops with more number of epiphytic bacteria in different raw silage ma- LAB are more suitable for natural silage (Lin et al. terials were quite variable in the present study. 1992). Thus, corn may be a better source of natural sil- The diversity and richness of the epiphytic bacterial age than alfalfa. community of alfalfa were significantly higher than those There are several undesirable microorganisms, such as of corn. The Shannon diversity index of corn and alfalfa anaerobic bacilli of the genus Clostridium, aerobic bac- was between 5 and 9, higher than those reported by teria of the genus Bacillus, coliform bacilli, in the fer- Drouin et al. (2019). The Shannon diversity index was mentation process and silage quality (Fabiszewska et al. higher for the fresh corn samples, with a mean of 5.26 ± 2019). We observed that Duobao No. 3 and Shengrui Wu et al. Annals of Microbiology (2021) 71:38 Page 9 of 11 Fig. 7 Genus level composition heat map for species clustering. Heat map is color-coded based on row z-scores. Colors range from bright red (strong positive correlation; i.e., r = 6) to bright blue (strong negative correlation; i.e., r = − 6). The red in the figure represents the genus with higher abundance, and the blue represents the genus with lower abundance in the 50 most abundant genera 565 in Hengshui have more Clostridium sensu stricto 1, natural silage. The results showed that the number of while Bacillus was mainly observed in SR4030 and Saidi bacteria belonging to Firmicutes, Bacilli, Lactobasubcil- 5 in Xingtai. Tao et al. used Illumina Miseq high- lales, Lactobacillaceae, Pediococcus, and Lactobacillus in- throughput sequencing technology to analyze the change creased, while Proteobacteria and Enterobacteriace in microflora structure in corn stalk before and after decreased (Tao and Diao 2016). Moreover, it was Fig. 8 Genus heat map of top 20 importance Wu et al. Annals of Microbiology (2021) 71:38 Page 10 of 11 revealed that the aerobic stability was increased by 66 ~ Author details School of Life Sciences, Hebei University, Baoding 071002, People’s Republic 312 h after the quantity of Clostridium in silage de- of China. Key Laboratory of Microbial Diversity Research and Application of creased in the aerobic stability test of silage (Jatkauskas Hebei Province, Engineering Laboratory of Microbial Breeding and and Vrotniakiene 2013). Preservation of Hebei Province, Institute of Life Sciences and Green Development, Baoding 071002, People’s Republic of China. Hebei Research Epiphytic bacteria on crops run throughout the whole Center for Geoanalysis, No. 180 Wusi East Road, Baoding City 071002, Hebei fermentation process, affecting the quality of the natural Province, People’s Republic of China. silage. These bacterial communities also have a succes- Received: 26 May 2021 Accepted: 28 August 2021 sion process, indicating that the microorganism on the forage greatly affect the quality of the natural silage. However, the structure of silage microbial community References and its mechanism of succession are still unclear, and Ali N, Wang SR, Zhao J, Dong ZH, Li JF, Nazar M, Shao T (2020) Microbial diversity more information is needed to reveal this complex fer- and fermentation profile of red clover silage inoculated with reconstituted mentation process (Xu et al. 2017). indigenous and exogenous epiphytic microbiota. Bioresour Technol 10: Bai Y (2011) Studies on the foliar microflora of bamboo forests in Sichuan. 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Biomed Res Int 2014:986048 Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Study on the diversity of epiphytic bacteria on corn and alfalfa using Illumina MiSeq/NovaSeq high-throughput sequencing system

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Abstract

Purpose: To investigate the diversity of the epiphytic bacteria on corn (Zea mays) and alfalfa (Medicago sativa) collected in Hengshui City and Xingtai City, Hebei Province, China, and explore crops suitable for natural silage. Methods: The Illumina MiSeq/NovaSeq high-throughput sequencing system was used to conduct paired-end sequencing of the community DNA fragments from the surface of corn and alfalfa collected in Hengshui and Xingtai. QIIME2 and R software were used to sort and calculate the number of sequences and taxonomic units for each sample. Thereafter, the alpha and beta diversity indices at of species level were calculated, and the abundance and distribution of taxa were analyzed and compared between samples. Result: At phylum level, the dominant groups were Proteobacteria (70%), Firmicutes (13%), Actinobacteria (9%), and Bacteroidetes (7%). Meanwhile, the dominant genera were Pseudomonas (8%), Acinetobacter (4%), Chryseobacterium (3%), and Hymenobacter (1%). Enterobacteriaceae (24%) were the most predominant bacteria in both the corn and alfalfa samples. Alpha diversity analysis and beta diversity indices revealed that the diversity of epiphytic microbial communities was significantly affected by plant species but not by region. The diversity and richness of the epiphytic bacterial community of alfalfa were significantly higher than those of corn. Conclusion: This study contributes to the expanding knowledge on the diversity of epiphytic bacteria in corn and alfalfa silage and provides a basis for the selection of raw materials. Keywords: Bacterial diversity, Alfalfa, Corn Introduction facilitates animal digestion and absorption, increases the Natural silage is the process of converting the fermenta- value of forage utilization, expands the source of forage, tion substrate (soluble sugar) in raw materials into acidic and adjusts the forage supply period (Shang et al. 2019). products, such as lactic acid through the proliferation of After silage, the nutrients will not be reduced. Silage also lactic acid bacteria (LAB) on crops. This process creates has an aromatic and sour taste, which stimulates the ap- an acidic environment and inhibits the proliferation of petite of livestock and increases feed intake. The quality harmful microorganisms, thereby preserving the nutri- of natural silage was greatly affected by the epiphytic tional content of raw materials (Zhang et al. 2011). As a microflora on crops. The fermentation time of natural storage technology, silage reduces forage nutrient loss, silage is very long. LAB cannot form dominant bacteria in a short time. Therefore, the crops with high LAB and * Correspondence: liuguixia1971@163.com; tangshude1234@163.com low epiphytic microbial diversity should be more suit- School of Life Sciences, Hebei University, Baoding 071002, People’s Republic able for natural silage. of China Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Wu et al. Annals of Microbiology (2021) 71:38 Page 2 of 11 Studies have observed that LAB, such as Lactobacillus, content was approximately 50 ~ 60%. The specific sam- Lactococcus, Leuconstoc, Streptococcus, Pediococcus, and pling results are presented in Table 1. Enterococcus, play a key role during the silage fermenta- tion processing (Gharechahi et al. 2017). The abundance Sample DNA extraction and PCR amplification, of LAB on crops determines the success of the silage quantification, pooling and sequencing process. Depending on the production of metabolites, DNA was extracted using the E.Z.N.A.® Soil DNA Kit LAB can be divided into two groups: homofermentative (Omega Bio-tek, Norcross, GA, USA) according to LAB and heterofermentative LAB. Homofermentative the manufacturer’s protocols, and quantified using LAB can produce more lactic acid, which can improve Nanodrop. The quality of DNA extracted was ob- the fermentation quality of silage. Moreover, they pro- served through electrophoresis on a 1.2% agarose gel. duce volatile fatty acids to inhibit the growth of aerobic The variable region of the 16S rRNA gene (single or bacteria and improve the aerobic stability of silage. consecutive multiple) or specific gene fragments was The epiphytic microflora greatly affects the quality of amplified using polymerase chain reaction (PCR). natural silage fermentation. And the fermentation qual- Subsequently, the PCR products were purified using ity of silage can be affected by different crops or even Vazyme VAHTSTM DNA Clean Beads and quantified the same crop grown under different environments con- fluorometrically. Sequencing libraries were prepared ditions (Ali et al. 2020; Huang et al. 2019). Choosing the using Illumina TruSeq Nano DNA LT Library Prep appropriate forage ingredients can help improve the Kit. PCR products that met the minimum concentra- quality of the silage. Corn (Zea mays) and alfalfa (Medi- tion required for analysis were electrophoresed in 2% cago sativa) are often utilized to produce silage. Identify- agarose gel to check for the correct size of the target ing the epiphytic microflora on forage can provide a bands. High-throughput sequencing was conducted scientific basis for effectively regulating the fermentation using Illumina Miseq/NovaSeq at Personalbio Com- process of silage. The population of eubacteria on corn pany (Shanghai, China). samples collected immediately was mainly composed of genera belonging to Proteobacteria (56.4 ± 1.5%), specif- Bioinformatics and statistical analysis ically to orders Pseudomonadales, Xanthomonadales, Bioinformatics was mainly performed using QIIME2 and Enterobacteriales; and Bacteriodetes (37.4 ± 1.7%), (2019.4) (Bolyen et al. 2018). Sequences were denoised specifically to orders Sphingobacteriales and Flavobac- using the DADA2 plugin (Callahan et al. 2016). Instead teriales (Drouin et al. 2019). Lactobacillales were of clustering by similarity, DADA2 only performs dere- substantial contributors of Firmicutes, with Leuconosto- plication or clustering at 100% similarity. Because caceae representing between 60% and 100% of the fresh DADA2 has not yet been adapted to all amplicons, we forage sample composition (Drouin et al. 2019). Entero- retained the OTU clustering-based Vsearch (Rognes bacteriaceae are predominant on corn and alfalfa (Lin et al. 2016) as an alternative. The abundance, distribu- et al. 1992). Yeasts and moulds are also major epi- tion, alpha, and beta diversity indices were analyzed phytic microorganisms on both crops (Lin et al. 1992; using QIIME2 and R software (Xie et al. 2016). The ac- Zhang 2011). However, the abundance of LAB on the cession numbers for sequencing data presented is raw material for ensiling is far less than that of aerobic PRJNA745034. bacteria, Escherichia coli, yeast, mould, and other harmful microorganisms (Zhang et al. 2011). The number of Results LAB on the surface of corn was greater than that of Species composition analysis other raw materials (Cai et al. 1999; Kasmaei et al. The specific composition of the microbial communities 2017). However, there are few reports on the epiphytic at each classification level in each sample was obtained microorganisms on corn and alfalfa. This study discussed using the statistics of the amplicon sequence variant the species and diversity of epiphytic bacteria on corn (ASV) table after rarefaction. The ggplot2 package in R and alfalfa. (Ginestet 2011) was used to plot the data and generate a histogram to visualize the number of taxa at each classi- fication level in the samples (Fig. 1). A microbial classifi- Materials and methods cation hierarchy tree was also generated to reveal the Collection of samples composition of all taxa at the same time using R lan- Samples were collected in the budding to the early flow- guage ggtree package (Yu et al. 2017). ering stage of alfalfa, and in the late milking to the early At phylum level, Proteobacteria (70%), Firmicutes waxing stage of corn. Alfalfa and corn samples were col- (13%), Actinobacteria (9%), and Bacteroidetes (7%) were lected in Xingtai (113°52′ E, 36°50′ N) and Hengshui the most dominant (Fig. 2). The four phyla have more (115°10′ E, 37°04′ N), Hebei Province. The moisture branches in the tree, which indicates that the genotypes Wu et al. Annals of Microbiology (2021) 71:38 Page 3 of 11 Table 1 Origin and grouping of microbial samples Places Plants Breed Serial number Groups Xingtai Corn Jinchu 100 D1 D1-1 D1-2 D1-3 Duobao No. 3 D2 D2-1 D2-2 D2-3 Shengrui 565 D3 D3-1 D3-2 D3-3 Yawangqingchu No. 8 D4 D4-1 D4-2 D4-3 Alfalfa SR4030 E1 E1-1 E1-2 E1-3 Saidi 5 E2 E2-1 E2-2 E2-3 Zhongmu No. 1 E3 E3-1 E3-2 E3-3 Hengshui Corn Duobao No. 3 F1 F1-1 F1-2 F1-3 Shengrui 565 F2 F2-1 F2-2 F2-3 Jinchu 100 F3 F3-1 F3-2 F3-3 Yawangqingchu No. 8 F5 F5-1 F5-2 F5-3 Alfalfa SR4030 G1 G1-1 G1-2 G1-3 Zhongmu No. 1 G2 G2-1 G2-2 G2-3 Saidi 5 G5 G5-1 G5-2 G5-3 of the dominant bacteria in the samples are diversified Microbial diversity analysis of corn and alfalfa samples in evolutionary relations. Interestingly, Lactobacillales Alpha diversity analysis was more abundant in corn samples than in alfalfa sam- Alpha diversity represents the diversity of species within ples (Fig. 2). Enterobacteriaceae (24%), which belongs to a habitat. Chao1 species index measures the species rich- Proteobacteria, was the most predominant family in both ness within a community (Chao 1984), while Shannon corn and alfalfa samples. Four genera have more than and Simpson indices measure the species diversity 1% of the reads and they are Pseudomonas (8%), Acineto- within a community (Simpson 1949). Observed species bacter (4%), Chryseobacterium (3%), and Hymenobacter indices measure community richness. Phylogenetic di- (1%) (Figs. 2 and 3). versity (PD) index represents diversity based on Fig. 1 The number of taxa at each classification level for different samples Wu et al. Annals of Microbiology (2021) 71:38 Page 4 of 11 Fig. 2 The pie chart (threshold 0.5%) of each branch node in the classification tree shows the proportion of the taxon in each group. The larger the sector area, the higher the abundance of the taxon in the group. The percentage above the taxon represents the percentage of the total bacteria evolution (Faith 1992). Meanwhile, Pielou’s evenness 1958). Results were plotted into a boxplot using the R to index represents species evenness (Pielou 1996) and exhibit the differences in diversity indices observed be- Good’s coverage index represents the coverage (Good tween different groups (Fig. 4). The microbial Wu et al. Annals of Microbiology (2021) 71:38 Page 5 of 11 Fig. 3 Histogram of genus level species composition community richness, diversity, evenness, and evolution- were dispersed. Samples of corn (Fig. 5D, F) were similar ary diversity of alfalfa (Fig. 4E, G) were higher than that and the samples of alfalfa (Fig. 5E, G) were similar. The of corn (Fig. 4D, F). However, the coverage of species in results showed plant species affected the epiphytic bac- the microbial community of alfalfa was lower than that terial communities, as compared with region. of corn. The microflora of alfalfa and corn collected in Xingtai had significant differences in community rich- Species difference analysis and biomarker ness and evolutionary diversity. Meanwhile, the micro- The number of ASV in groups D, E, F, and G was 6683, flora of alfalfa and corn collected in Hengshui had highly 8305, 6920, and 8080 respectively (Fig. 6). There are 545 significant differences in community richness, diversity, ASVs in common, accounting for 2.34% of the total and evenness. Thus, the diversity of epiphytic bacterial ASV. community is significantly affected by plant species. All We utilized the relative abundances of the top 50 gen- alpha diversity indices in the microflora of alfalfa col- era to generate a heat map by heatmap package in R lected in both sites were not significantly different. (Zhao et al. 2014). Heatmap shows a data matrix where Alpha diversity indices, excluding evolutionary diversity, coloring gives an overview of the numeric differences. In in the microflora of corn collected in Xingtai and Heng- the genus-level species composition heat map for species shui were not significantly different. We can conclude clustering, red and blue patches indicate that the genera that the region has no significant effect on the diversity are more abundant and less abundant in a sample than of epiphytic microbial communities. the other sample. Lactic acid bacteria, such as those be- longing in the genera Leuconostoc and Lactobacillus, Beta diversity analysis have an important effect on silage fermentation and are The microbial communities in alfalfa and corn samples among the top 50 genera in terms of relative abundance. were compared using non-metric multidimensional scal- Leuconostoc was mainly present in groups D2, F1, F2, ing (NMDS) based on the weighted UniFrac distance and F3. Lactobacillus was mainly present in groups D1, (Lozupone and Knight 2005). Each point in the diagram D3, D4, F1, F2, F3, E1, E2, and E3 (Fig. 7). As presented represents a sample, and the different colored dots indi- in Fig. 7, groups F1 and F2 had higher abundance of cated different samples (Fig. 5). Samples were clustered Clostridium sensu stricto 1, which is harmful to according to their similarity, and the closer the distance fermentation. between two points is, the more similar the two samples We obtained the distribution of important species in are. Alfalfa group samples aggregated in the NMDS ana- each group by using the algorithm analysis of random lysis diagram, while the samples from the corn group forests (Breiman 2001) (Fig. 8). The abscissa represented Wu et al. Annals of Microbiology (2021) 71:38 Page 6 of 11 Fig. 4 Grouping box plot of alpha diversity index. The two groups below the two ends of the horizontal line indicate that the index of two groups are significant different. The number under the diversity index label is the P value of the Kruskal-Wallis test the importance of species to the classifier model, and Spirosoma, Hymenobacter, Bacillus, Actinomycetospora, the ordinate represented the taxon name at genus level. Taibaiella, and Sphingobacterium, can be considered The importance of genus in shaping the bacterial com- markers of differences in these groups. Most of these munity in each group decreases successively. These genera belong to Proteobacteria and Bacteroidetes. How- highly important genera, namely Pedobacter, Nocar- ever, LAB, which have a positive impact on fermentation dioides, Chryseobacterium, Burkholderia−Caballeronia were not observed. The presence of Bacillus is worth −Paraburkholderia, Paracoccus, Pseudomonas, Acineto- noting. Bacillus causes the silage to deteriorate, leading bacter, Allorhizobium−Neorhizobium−Pararhizobium to a rotten and smelly product. Bacillus was mainly ob- −Rhizobium, Larkinella, Mucilaginibacter, Sphingomo- served SR4030 and Saidi 5 in Xingtai. We should choose nas, Brevundimonas, Siphonobacter, Methylobacterium, crops with more LAB, less Bacillus and Clostridium, and Wu et al. Annals of Microbiology (2021) 71:38 Page 7 of 11 Fig. 5 NMDS analysis based on the weighted UniFrac distance lower epiphytic microbial diversity for natural silage, be- Drouin et al. (2019) observed that the populations of cause LAB on these crops may form dominant bacteria bacteria on the corn samples collected immediately after in a short time, reducing the loss of nutrients. Taking inoculation, but prior to ensiling, were mainly composed these factors into consideration, the epiphytic bacteria of genera belonging to Proteobacteria (56.4 ± 1.5%), spe- on Duobao No. 3 in Xingtai and Jinchu 100 in Hengshui cifically in orders Pseudomonadales, Xanthomonadales, may be better for natural silage. However, the content of and Enterobacteriales; and to Bacteriodetes (37.4 ± moisture, protein, and sugar of plants also influence the 1.7%), specifically in orders Sphingobacteriales and Fla- quality of silage. We are going to do further study to vobacteriales. In another study, 89.6% of the bacterial choose the crops that are suitable for natural silage. 16S rRNA gene sequences were associated with the phylum Proteobacteria, and 8.1% were associated with Discussion the phylum Firmicutes; other phyla identified were Acti- Corn and alfalfa are widely used raw materials for nat- nobacteria (0.2%) and Bacteroidetes (0.2%), before alfalfa ural silage. The epiphytic bacteria on crops substantially ensiling (McGarvey et al. 2013). The populations of bac- affect the quality of natural silage. Identifying the epi- teria in the fresh corn and alfalfa samples in previous phytic microflora on forage can provide a scientific basis studies were mainly composed of genera belonging to for effectively regulating the fermentation process of nat- Proteobacteria, Firmicutes, Actinobacteria, and Bacteroi- ural silage. There are only few detailed reports on the detes, although their abundance varied slightly (Ali et al. epiphytic microorganisms of corn and alfalfa. Most stud- 2020; Drouin et al. 2019; McGarvey et al. 2013). Most of ies reported the microbes using the plate count method the epiphytic bacteria of corn and alfalfa belong to Pro- (Cai et al. 1999; Lin et al. 1992). Drouin et al. (2019) teobacteria, and Enterobacteriaceae are the most pre- studied the epiphytic microflora on corn using high- dominant bacterial family on corn and alfalfa (Lin et al. throughput sequencing. However, these studies are not 1992). These results are consistent with the present sufficiently detailed. In this study, the Illumina MiSeq/ study. Proteobacteria was the most prevalent phylum in NovaSeq system was used to analyze the diversity of epi- fresh corn, and the bacterial community of alfalfa was phytic bacteria on corn and alfalfa. highly dominated by Firmicutes during the ensiling Wu et al. Annals of Microbiology (2021) 71:38 Page 8 of 11 Fig. 6 Venn diagram of sample (group) ASV period, when the aerobic environment was changed to 0.27, in the previous study (Drouin et al. 2019). We ob- anaerobic. Moreover, the abundance of Firmicutes in- served LAB, such as Leuconostoc and Lactobacillus,in creased significantly (Drouin et al. 2019). Sequences af- the top 50 genera in average abundance. Several studies filiated with Lactobacillales were substantial contributors have shown that corn has higher LAB composition than to the Firmicutes phylum order (Drouin et al. 2019). In other crops. For example, the number of LAB on the our study, the main epiphytic bacterial composition in surface of corn was twice than that of sorghum and al- corn and alfalfa were consistent with the results of previ- falfa, and 20 times than that of ryegrass (Cai et al. 1999). ous studies. Meanwhile, the total number of LAB on corn is seven Our results suggest that the diversity of epiphytic bac- times than that of grass and 15 times than that of clover terial communities is not affected by region, but it is sig- (Kasmaei et al. 2017). The lactic acid and acetic acid nificantly affected by plant species. This may also be contents of silage corn silage corn, elephant grass, and associated with the similar environments of the two sugarcane tops were significantly increased by adding sites; thus, environment cannot affect the epiphytic mi- the epiphytic microorganisms of corn straw, and the aer- croorganisms of the plants. However, the epiphytic mi- obic stability of elephant grass silage was positively af- croorganisms of forage are affected by forage species, fected (Huang et al. 2020). Kasmaei et al. (2017) stage of maturity, weather, mowing, field-wilting, chop- observed that the increase in lactic acid and acetic acid ping process humidity, solar radiation, plant surface content in corn straw silage by epiphytic microorgan- structure, and plant nutrient distribution (Lin et al. isms may be related to the abundance of Lactococcus 1992; Bai 2011). This may be the reason the species and and Leuconostoc (Kasmaei et al. 2017). Crops with more number of epiphytic bacteria in different raw silage ma- LAB are more suitable for natural silage (Lin et al. terials were quite variable in the present study. 1992). Thus, corn may be a better source of natural sil- The diversity and richness of the epiphytic bacterial age than alfalfa. community of alfalfa were significantly higher than those There are several undesirable microorganisms, such as of corn. The Shannon diversity index of corn and alfalfa anaerobic bacilli of the genus Clostridium, aerobic bac- was between 5 and 9, higher than those reported by teria of the genus Bacillus, coliform bacilli, in the fer- Drouin et al. (2019). The Shannon diversity index was mentation process and silage quality (Fabiszewska et al. higher for the fresh corn samples, with a mean of 5.26 ± 2019). We observed that Duobao No. 3 and Shengrui Wu et al. Annals of Microbiology (2021) 71:38 Page 9 of 11 Fig. 7 Genus level composition heat map for species clustering. Heat map is color-coded based on row z-scores. Colors range from bright red (strong positive correlation; i.e., r = 6) to bright blue (strong negative correlation; i.e., r = − 6). The red in the figure represents the genus with higher abundance, and the blue represents the genus with lower abundance in the 50 most abundant genera 565 in Hengshui have more Clostridium sensu stricto 1, natural silage. The results showed that the number of while Bacillus was mainly observed in SR4030 and Saidi bacteria belonging to Firmicutes, Bacilli, Lactobasubcil- 5 in Xingtai. Tao et al. used Illumina Miseq high- lales, Lactobacillaceae, Pediococcus, and Lactobacillus in- throughput sequencing technology to analyze the change creased, while Proteobacteria and Enterobacteriace in microflora structure in corn stalk before and after decreased (Tao and Diao 2016). Moreover, it was Fig. 8 Genus heat map of top 20 importance Wu et al. Annals of Microbiology (2021) 71:38 Page 10 of 11 revealed that the aerobic stability was increased by 66 ~ Author details School of Life Sciences, Hebei University, Baoding 071002, People’s Republic 312 h after the quantity of Clostridium in silage de- of China. Key Laboratory of Microbial Diversity Research and Application of creased in the aerobic stability test of silage (Jatkauskas Hebei Province, Engineering Laboratory of Microbial Breeding and and Vrotniakiene 2013). Preservation of Hebei Province, Institute of Life Sciences and Green Development, Baoding 071002, People’s Republic of China. Hebei Research Epiphytic bacteria on crops run throughout the whole Center for Geoanalysis, No. 180 Wusi East Road, Baoding City 071002, Hebei fermentation process, affecting the quality of the natural Province, People’s Republic of China. silage. 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Biomed Res Int 2014:986048 Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Journal

Annals of MicrobiologySpringer Journals

Published: Dec 1, 2021

Keywords: Bacterial diversity; Alfalfa; Corn

References