Germination of pecan seeds changes the microbial community

Sampling procedure

Fresh pecan seeds were used in the present experiment, which were provided by the Jiang Tao Family Farm, Lai ’an County, Anhui Province, China in November 2022, and the cultivar is “Pawnee”. The seeds were kept at a 30 °C room temperature in Chuzhou University’s Plant Physiology Laboratory. For ten days, pecan seeds were imbibed at room temperature. The seeds were then placed at a germination box covered with moist absorbent cotton and placed in a 30 °C constant temperature in full light incubator. The samples were taken four times: S1 (imbibed 10 days), S2 (after 5 days in the incubator), S3 (seed endocarp break) and S4 (seed radicle protrusion) during seed germination. At each stage of germination, we collected 5–10 seeds that germinated consistently and surface sterilized them with 2% NaClO (sodium hypochlorite) by shaking them for 4 min. In order to eliminate residues of NaClO that may interfere with the next steps, the seeds were rinsed five times in sterile MilliQ water. Finally, all samples were immediately frozen in liquid nitrogen. For microbial sequencing, the samples were stored at −80 °C in a refrigerator.

DNA extraction and amplification

CTAB (cetyl trimethylammonium bromide) technique was used to extracted total genome DNA from seed samples (Niemi et al., 2001). We have evaluated DNA content and purity of 1% agarose gels, what’s more, depending on the concentration, DNA was diluted to 1 ng·μL⁻¹ with sterile water. To generate the bacterial libraries, we used the 799F (5′-AACMGGATTAGATACCCKG-3′) and 1193R (5′-ACGTCATCCCCACCTTCC-3′) primers set with the unique 6-nt barcode at 5′ of the forward primer to amplify the V5–V7 region of the 16S rRNA gene for each sample. The construction of the fungal libraries was similar to the bacterial libraries, except that they were amplified using ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) for the ITS2 region. All PCR reactions were performed using Phusion^® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA). One-load buffer (including SYB green) was mixed with PCR products at a 1:1 volume ratio, and electrophoretic detection was performed on 2% agarose gels. The PCR products were mixed in an equal density ratio. The mixed PCR products were then purified using the Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany). In accordance with the manufacturer’s recommendations, sequencing libraries were generated using the Truseq^® DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA). Index codes were also added to the libraries. A Qubit@ 2.0 Fluorometer (Thermo Scientific, Waltham, MA, USA) and an Agilent Bioanalyzer 2100 system were used to evaluate the quality of the library. An Illumina MiSeq PE300 platform was used to sequence the libraries and generate paired reads.

DNA sequence analysis

All raw data from the 16S V5–V7 bacterial region and the fungal ITS1 region was processed by QIIME for quality-controlled process (V1.9.1, http://qiime.org/), and FLASH for paired reads (V1.2.7, http://ccb.jhu.edu/software/FLASH/) (Bolyen et al., 2019; Magoč & Salzberg, 2011). For bacteria and fungi, annotation was done by matching the Silva sequences with the UCHIME algorithm and Unite database (ITS: http://unite.ut.ee/) (UCHIME, https://www.drive5.com/usearch/manual/uchime_algo.html) (Koljalg et al., 2013; Quast et al., 2012).

Statistical analysis

For the analysis of Alpha Diversity, four indices of species diversity are applied, including Observed Species, Chao1, Shannon, and Simpson. These indices were calculated using QIIME (Version 1.9.1) and visualized using R package ggplot2 (Version 2.15.3). One-way ANOVA was used to determine significant differences at a P-value of 0.05; if significant differences were detected, Duncan’s post hoc test was used to identify the values that differed from the others. A beta diversity analysis was used to determine the differences in species complexity among samples, and beta diversity on Bray-Curtis was calculated using QIIME software (version 1.9.1).

Alpha and beta-diversity of pecan seed microbial communities

Pecan seeds were sequenced using the Illumina MiSeq high-throughput platform to determine the diversity of bacterial and fungal communities and their ASVs. The Good’s coverage index of all samples was greater than 98.5%, indicating that the sequencing results could accurately reflect the true status of the bacterial and fungal communities within the samples. In terms of Shannon diversity index, bacteria had a higher diversity at S3 (seed endocarp break) stage of seed germination, whereas fungi had a higher diversity at S1 (imbibed 10 days) stage. Both the ACE index and the Chao1 index of seed endophyte microorganisms in the four stages had the same trend, and at S3 stage had the highest, indicating the most abundant species in the S3 stage. Additionally, the species richness at the S4 (seed radicle protrusion) stage in fungi was significantly lower than that of the other samples. It was found that the Pielou evenness index of bacteria was generally small, indicating that the species distribution of bacteria in pecan seeds was not uniform. However, the fungal index is close to one, indicating that fungal species are equally distributed, and at the S1 stage, species distribution is the most uniform (Table 1).

Principal components analysis (PCA) is a two-dimension reduction ordination analysis method based on Bray–Curtis distance for assessing the similarities and differences between bacterial and fungal communities (Fig. 1). In order to investigate the separation of different germination process, we performed PCA in our study. In bacterial PCA, 36.49% of PC1 and 20% of the total variance were explained by PC2 (Fig. 1A). In fungal PCA, PC1 accounted for 36.01% and PC2 for 23.24% of the overall variance (Fig. 1B).

Microbial community composition in pecan seed germination process

According to the analysis of ASV annotation results, different germination stages of pecan seeds bacteria include 12 phyla, 18 classes, 38 orders, 60 families, 82 genera and 62 species (Fig. 2). Among all sequences, the dominant bacterial phyla (relative abundance >1%) were Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Acidobacteria, Euryarchaeota, Deniococcus-Thermus, Spirochaetes, Ignavibacteriae, Parcubacteria. It can be seen from Figs. 2A–2E that the endophytic bacteria in the seeds at the S3 and S4 stages are different from those in the previous two stages. At the phylum level, the two main phylums have undergone major changes, and Cyanobacteria in the first two stages. The relative abundance of the first stage is relatively higher, and in the latter two stages, the relative abundance of Proteobacteria increases, while the abundance of Cyanobacteria decreases. The S3 and S4 stages represent two important stages of seed germination, respectively, and they require different metabolites at the bacterial community level. So, the present study was found that in these two stages, their bacterial communities at different levels have great differences and changes.

However, the changes in the fungal relative abundance of the major families during germination are shown in Fig. 3. Pecan seeds fungi include seven phyla, 14 classes, 23 orders, 30 families, 35 genera and 22 species. Among all sequences, the dominant fungal phyla (relative abundance >1%) were Anthophyta, Ascomycota, Basidiomycota, Cercozoa, Bacteroidetes, Chlorophyta, Chytridiomycota, and Mortierellomycota. Among other species, we observed Juglandaceae, an endophyte closely related to pecan.

Core and specific microbiome of pecan seeds

In order to assess the relationship between bacterial and fungal communities, the ASVs shared by different germination stages of pecan seeds were presented as an UpSet plot in Figs. 4A and 4B. UpSet plots are used for visualizing the number and overlap of different ASVs. As shown in Fig. 4A, all samples shared the majority of bacterial ASVs. There were 40 ASVs shared by S1, S3, and S4, 17 ASVs shared by S1 and S3 and 24 ASVs shared by S2 and S4. S3 stage was found to have the most unique ASVs compared with the other three stages. In all four germination stages, a total of 180 fungal ASVs was found. 64 ASVs (35.6% of the total ASVs) were shared between them (Fig. 4B) and the S3 stage shared highest number of fungal ASVs (37 OTUs, which is 20.6% of the total ASVs). To further analyze the core microbiome of the pecan seeds on the phylum level, the Venn network diagram was used because it can be more clearly to evaluate the distribution of ASVs among different stages. The common core bacterial microbiome of pecan seed consisted of members of Cyanobacteria, Proteobacteria, and Actinobacteria (Fig. 4C). But in fungal communities, the common core microbiome is Anthophyta and Ascomycota (Fig. 4D).

To further explore the effects of different germination stages of pecan seed on microbial communities, we analyzed bacterial and fungal communities using samples subjected to S1, S3 and S4 treatments, which affected bacterial and fungal diversity most strongly. The ternary plot showed that high-abundance ASVs belonging to Proteobacteria and Actinobacteria phyla were detected upon S3 and S4 treatments, but the relative abundances of these ASVs in the S1 were extremely low. Moreover, comparing the relative abundances of most ASVs in S3 and S4 stages, S3 is lower than S4 (Fig. 5A). Then, samples treated with S1, S3 and S4 were used to further study the fungal community changes. In the fungal community, ASVs from Ascomycota phylum were observed to have a high abundance in the S3 and S4 treatments, but the relative abundance of most ASVs in the S1 group was lower (Fig. 5B).

Biomarkers changes in the relative abundances of different germination stages in pecan seeds

In study, linear discriminant analysis (LDA) and effect size (LEfSe) analysis were conducted to identify bacterial and fungal communities as biomarkers (Fig. 6). Compared to the seed bacterial community (two classes, five orders, six families, nine genera and nine species), and we found significantly richer biomarker species in the fungal communities (five classes, four orders, nine families, 10 genera and 12 species) of pecan seeds in different germination stages. All taxonomic levels of seed bacteria were associated with 10 taxa derived from S2, 29 taxa derived from S3, and 14 taxa derived from S4. Among them, Proteobacteria, Gamma proteobacteria and Cyanobacteria were identified as highly abundant biomarkers in the bacterial community. Among all taxonomic levels of seed fungi, 28 biomarkers were related to S1 and 18 were related to S2, 43 biomarkers were related to S3, 12 biomarkers related to S4 germination stage. Ascomycota and Sordariomycetes were significantly abundant in the seed fungal community.