Study on the Diversity of Epiphytic Bacteria on Corn and Alfalfa using Illumina MiSeq/NovaSeq High-throughput Sequencing System

Purpose To investigate the diversity of the epiphytic bacteria on corn and alfalfa collected in Hengshui city and city, province, China. Methods Illumina MiSeq/NovaSeq High-throughput sequencing systerm was used to conduct Paired-end sequencing of community DNA fragments from surface of corn and alfalfa in Hengshui and Xingtai. QIIME2 and R language were employed to sort and calculate the number of sequences and taxonomic units for each sample. Thereafter, the abundance, distribution, alpha diversity index of species, beta diversity and the differences of abundance among the samples were analyzed. Result Enterobacteriaceae are the most predominant bacteria on both corn and alfalfa samples. Alpha diversity analysis and beta diversity analysis showed that the diversity of epiphytic microbial community was signicantly affected by plant species, but not by region. The diversity and richness of epiphytic bacterial community of alfalfa were signicantly higher than that of corn, yet corn had more LAB than alfalfa samples.


Introduction
Silage is the process of converting the fermentation substrate (soluble sugar) in raw materials into acidic materials such as lactic acid through the proliferation of lactic acid bacteria, creating an acidic environment and inhibiting the proliferation of harmful microorganisms, thereby preserving the nutritional content of raw materials (Zhang et al. 2011). As a silage storage technology, silage has the effects of reducing forage nutrient loss, facilitating animal digestion and absorption, increasing the value of forage utilization, expanding the source of forage, and adjusting the forage supply period (Shang et al. 2019).
After silage, the nutrients will not be reduced. It also has an aromatic and sour taste, which stimulates the appetite of livestock and increases the feed intake.
Studies show that Lactic acid bacteria (LAB) play a key role during the silage fermentation processing, such as Lactobacillus, Lactococcus, Leuconstoc, Streptococcus, Pediococcus and Enterococcus (Gharechahi et al. 2017). The number of LAB on crops determines the success of silage process.
According to the different metabolites of lactic acid bacteria, they can be divided into homomorphic lactic acid bacteria and heteromorphic lactic acid bacteria Homogeneous lactic acid bacteria can produce more lactic acid, which can improve the fermentation quality of silage. The heteromorphic lactic acid bacteria can produce lactic acid and volatile fatty acids to inhibit the growth of aerobic bacteria and improve the aerobic stability of silage.
The epiphytic micro ora greatly affect the fermentation quality of silage. And the fermentation quality of

Collection of Samples
When alfalfa was in the budding stage to the early owering stage, and when the corn silage material was in the late milking stage to the early waxing stage, alfalfa and corn were collected in Xingtai and Hengshui, Hebei Province. And the moisture content of them was about 50 ~ 60 %. The speci c sampling results are shown in Table 1. Table 1 Origin and grouping of microbial samples.

Places
Plants Breed Serial number Groups

Sample DNA Extraction and PCR Ampli cation, Quanti cation, Pooling and Sequencing
The DNA was extracted and quanti ed with Nanodrop. DNA extraction quality was detected using electrophoresis on a 1.2% agarose gel. And the variable region of rRNA gene (single or consecutive multiple) or speci c gene fragment could be ampli ed by PCR. Subsequently, the PCR products were puri ed using the Vazyme VAHTSTM DNA Clean Beads and quanti ed by uorescence. Sequencing libraries were prepared using Illumina's TruSeq Nano DNA LT Library Prep Kit. PCR products with appropriate concentration and the correct size of the target band were detected by 2% agarose gel electrophoresis. High-throughput sequencing was conducted using Illumina Miseq/NovaSeq. 3. Results

Species Composition Analysis
Through the statistics of the ASV/OTU table after pumping, the speci c composition table of microbial communities in each sample at each classi cation level can be obtained. Then we use the R script to plot the data in the table into a histogram (Fig. 1) to visually show the number of taxa at each classi cation level for different samples. In order to show the composition of all taxa at the same time, we draw a microbial classi cation hierarchy tree using ggtree in the R language.
The advantage bacterium group are Proteobacteria (70%), Firmicutes (13%), Actinobacteria (9%) and Bacteroidetes (7%) in all microbiome samples coming from corn and alfalfa in Hengshui and Xingtai (Fig. 2). The four dominant bacteria have more branches, which indicates that the genotypes of the dominant bacteria in the samples are diversi ed in evolutionary relations. Lactobacillales were found mainly in the samples of corn (Fig. 2). Enterobacteriaceae (24%) belonging to the Proteobacteria phylum are the most predominant bacteria on both corn and alfalfa samples. More than 1% of the reads from 4 genera belonging to Proteobacteria and Bacteroidetes including Pseudomonas (8%), Acinetobacter (4%), Chryseobacterium (3%) and Hymenobacter (1%) (Fig. 3).

Alpha diversity analysis
Alpha diversity represents the diversity of species within habitats. Chao1 (Chao. 1984) and Observed species indices measure community richness. Shannon and Simpson (Simpson. 1949) indices measure community diversity. Faith's PD (Faith., 1992) index represents diversity based on evolution. Pielou's evenness (Pielou. 1966) index represents the evenness. Good's coverage (Good. 1958) index represents coverage. And the speci c results were plotted into a boxplot using the R script to visually show the difference of alpha diversity between different groups. It can be seen that the microbial community richness, diversity, evenness and evolutionary diversity of microbiome samples of alfalfa (group E and G) is higher than that of maize (group D and F) on average. However, the coverage of species in a community of microbiome samples of alfalfa is lower than that of maize (Fig. 4). The microbiome samples of alfalfa and maize in Xingtai have extreme signi cant differences in community richness and evolutionary diversity. And the microbiome samples of alfalfa and maize in Hengshui have highly signi cant differences in community richness, diversity and evenness. Thus, the diversity of epiphytic microorganism community is signi cantly affected by plant species. All alpha diversity indices of alfalfa in different areas have no signi cant difference. And alpha diversity indices of maize except the evolutionary diversity have no signi cant difference in Xingtai and Hengshui. We can conclude that the region has no signi cant effect on the diversity of epiphytic microbial community. The epiphytic microbial diversity of Shengrui 565 is lower than other breeds in the two places.

Beta diversity analysis
The microbial communities in alfalfa and maize samples were compared using NMDS based on the weighted UniFrac distance (Lozupone and Knight. 2005). Each point in the diagram represents a sample, and different colored dots indicate different samples (Fig. 5). Samples are clustered according to their similarity, and the closer the distance between two points is, the more similar the tow samples are. Alfalfa group samples were aggregated in the NMDS analysis diagram, while the samples of the maize group were dispersed. Samples of corn (group D and F) are similar and the samples of alfalfa (group E and G) are similar. The results showed that epiphytic bacteria were more affected by species than by region.

Species difference analysis and biomarker
The number of ASV/OUT in group D, E, F and G are 6683, 8305, 6920, 8080 respectively (Fig. 6). And there are 545 ASV/OUTs in common.
We use the abundance data of the top 50 genera in average abundance to make a heat map. In the genus-level species composition heat map for species clustering, red patches indicate that the genera are more abundant in this sample than other samples, and blue patches indicate that the genera are less abundant in this sample than other samples. Lactic acid bacteria have an important effect on the silage fermentation, such as Leuconostoc and Lactobacillus in the top 50 genera in average abundance. Leuconostoc mainly exist in group D2, F1, F2 and F3. Lactobacillus mainly exist in group D1, D3, D4, F1, F2, F3, E1, E2 and E3 (Fig. 7). Group F1 and F2 have more Clostridium sensu stricto 1 that are harmful to fermentation (Fig. 7).
Through the algorithm analysis of Random Forests (Breiman. 2001), we obtained the distribution of important species in each group (Fig. 8). The abscissa is the importance of species to the classi er model, the ordinate is the taxon name at the level of genus from top to bottom, and the importance of species in in uencing grouping decreases successively. These highly important species can be considered markers of differences in these groups, and they are Pedobacter, Nocardioides, Chryseobacterium, Burkholderia − Caballeronia − Paraburkholderia, Paracoccus, Pseudomonas, Acinetobacter, Allorhizobium − Neorhizobium − Pararhizobium − Rhizobium, Larkinella, Mucilaginibacter, Sphingomonas, Brevundimonas, Siphonobacter, Methylobacterium, Spirosoma, Hymenobacter, Bacillus, Actinomycetospora, Taibaiella, Sphingobacterium. Bacillus mainly exist in SR4030 and Saidi 5 in Xingtai.

Discussion
The results show that the diversity of epiphytic microorganism community is not affected by region, yet it is signi cantly affected by plant species. It may also be because the environment of the two places is similar and it cannot affect the epiphytic microorganisms of the plants. The species and number of epiphytic microorganisms on different silage raw materials are quite variable. Epiphytic microorganisms of forage are affected by forage species, stage of maturity, weather, mowing, eld-wilting, the chopping process humidity, solar radiation, plant surface structure and plant nutrient distribution (Lin et al. 1992;Bai. 2011).
The diversity and richness of epiphytic bacterial community of alfalfa were signi cantly higher than that of corn, yet corn had more LAB than alfalfa samples. Many studies have shown that corn have more LAB than other crops. For example, the number of LAB on the surface of corn was twice that of sorghum and alfalfa, and 20 times that of ryegrass (Cai et al. 1999 Epiphytic bacteria of crops run through the whole fermentation process, affecting the quality of silage. These bacterial communities also have a succession process, indicating that the microorganism attached to the forage itself has a great impact on the quality of silage. However, the study of silage microbial community and its mechanism of succession is still unclear, and more information is needed to reveal this complex fermentation process (Xu et al. 2017).

Conclusion
In summary, the advantage bacterium group are Proteobacteria (70%), Firmicutes (13%), Actinobacteria (9%) and Bacteroidetes (7%) on corn and alfalfa in Xingtai and Hengshui. At the genus level, Pseudomonas (8%), Acinetobacter (4%), Chryseobacterium (3%), Hymenobacter (1%) were the main bacteria genera. This study showed that the diversity of epiphytic microbial community was signi cantly affected by species, but not by region. The composition richness and diversity of microbe of alfalfa are higher than that of maize in both Xingtai and Hengshui, yet corn have more LAB than alfalfa samples. Figure 1 The number of taxa at each classi cation level for different samples.