Microbiome structure of hemp
Microbiome diversity is defined as the number and abundance distribution of specific species of microbes44,45. Thus, the current research evaluated the microbial diversity and abundance of hemp species rich in CBD (Sweet Sensi and Cherry Wine) and Fiber (American Victory and Unknown). In this study, we evaluated both the rhizosphere (root and soil) and the phyllosphere (stem and leaf). The results from all samples showed that over 1,900,000 bacterial genera raw ASVs and over 4,200,000 fungal raw ASVs comprised 469 different genera. We found the fungal ASVs to be more abundant than bacterial ASVs. We also found that bacterial ASVs were significantly higher in rhizosphere soil (70,000) and root (47,000) when compared to phyllosphere stem and leaf (28,000–36,000) (Table S6). Interestingly, the overall fungal ASV abundance showed significantly higher counts across the phyllosphere than the rhizosphere. We found 118,000–131,000 ASVs in leaf and stem compartments and 74,000–81,000 counts in soil and root compartments. In the case of genotypes, a varying abundance of ASV count was observed across different compartments. Overall, the Cherry Wine showed significantly higher ASVs, followed by American Victory-1. We observed relatively high ASVs in the endospheric microbiome compared to the soil microbiome (Table S6).
We assessed the shared and unique ASVs across different genotypes and their compartments. The results showed that the bacterial ASVs were significantly shared in soil between the different genotypes, most likely due to their being grown in similar soil conditions. However, in the case of fungi, 14.6% more highly unique ASVs were found in fiber than in CBD-rich hemp species. Contrarily, a more varying distribution of ASVs was observed across different genotype compartments. The uniqueness and sharing of bacterial and fungal ASV recovered reduced from soil > root > stem > leaf compartments. More unique ASVs were retrieved from the CBD leaf (53.6%) than the fiber genotype (Fig. 2). The beta diversity data was subjected to PERMANOVA analysis, and interestingly, the stem compartment was significantly different (p < 0.05). This could be because the stem composition is different in CBD when compared to fiber-producing hemp varieties. Fiber-producing varieties tend to recruit different bacterial and fungal microbiomes than CBD-producing varieties. By digging into the CBD vs. fiber-producing varieties, the root compartment had significant (p < 0.05) variation in bacterial microbiomes. Likewise, the fungal microbiome was significantly (p < 0.05) different in the rhizosphere and stem compartment.
Upset plots showing the relationship between ASVs in plant genotypes based on the organ. Venn diagrams show the genotypes’ correlation when split into CBD or Fiber production. Yellow represents CBD-producing plants, while green represents Fiber-producing plants.
Rhizospheric and phyllosphere microbiome of hemp
In the soil, the most abundant (≥ 1% relative abundance) bacteria phyla were Firmicutes, Proteobacteria, Myxococcota, Actinobacteriota, Bacteroidota, Plantomycetota, Acidobacteriota, and Chloroflexi. Of the eight phyla in the data, the top 3 phyla were Proteobacteria, Acidobacteria, and Actinobacteria. Except for root parts, Firmicutes are significantly abundant compared to soil samples. Proteobacteria and Actinobacteria remain highly abundant in the root system. The top genera (≥ 1% relative abundance) are Streptomyces, Bacillus, and Pseudomonas across all genotypes. Among the keystone genera of American Victory-1 were from Family Kineosporiceae, which is roughly 15% relatively abundant, or genus Xylella, present at 16% relative abundance (Fig. 3).
Based on Genus level analysis, Taxaomic bar graphs show the relative abundance of bacteria. The graphs are split vertically by organ and horizontally by genotype.
In the stems and leaves of the plants, we found that Proteobacteria remain the top phylum, accounting for almost 97.5% of the Cherry Wine genotype. At the same time, Proteobacteria remains the top phylum for all other genotypes, between 60 and 98%. Firmicutes (0.5–21%) and Actinobacteria (1–14%) continue to play an important role in the stem. At the same time, Actinobacteria‘s presence appears to be reduced in the leaf of hemp, with the percent change being between 34 and 89% decrease. The top genera (≥ 1% relative abundance) for hemp leaves were Bacillus, Pseudomonas, Pantoea, Xylella, Sphingomonas, and Methylobacterium-Methylorubrum (Fig. 3).
The top fungal phylum abundant (≥ 1% relative abundance) in the soil were Ascomycota, Mortierellomycota, and unidentified organisms. In the root system, we found that Ascomycota remains highly abundant across all hemp genotypes and their organs, while Basidiomycota was abundant in genotype American Victory-1. For both the stem and leaves, the fungal phyla Ascomycota and Basidiomycota were the most prevalent phyla, with unidentified phyla still being present at very low abundance for all plant organs. Ascomycota is the most abundant phylum, with relative abundance being around 77–90% in the stem and leaves.
Many fungal genera were found throughout the plant genotypes. In the root fungal diversity, we found the six most abundant genera (≥ 1% presence): Gibberella, Fusarium, unidentified (Class: Sordariomycetes), Alternaria, Neocosmospora, and Septoria. These belong to Ascomycota. As expected, fewer genera were found from the phylum Basidiomycota. These genera were Athelia, and Rhizoctonia. We also found that unidentified organisms were abundant here. The top genera found in the stem and leaves of hemp genotypes appear to differ from that of the root system: Septoria, Cladosporium, Alternaria, Gibberella, and Moesziomyces (Fig. 4). The only genus Moesziomyces belonged to Basidiomycota, whereas most genera were Ascomycota.
Taxonomic bar graphs showing the relative abundance of fungi based on Genus level analysis. The graphs are split vertically by organ and horizontally by genotype.
Microbiome variation in genotype
When analyzing the Principal Coordinate Analysis (PCoA), the Bray–Curtis distance method was utilized alongside the Hellinger method for all ß-diversity testing. All soil communities are tightly grouped around the center of the PCoA graph, showing high relatedness in all samples. Sweet Sensi has the most significant distance from the rest in bacterial communities, while Cherry Wine has the largest distance in the fungal communities. Using PERMANOVA, both communities and all organs had significant variation. The leaf bacterial communities are roughly equivalent, with American Victory-1 and Sweet Sensi having the closest relatedness. Fungal communities in plant leaves, closely related to American Victory-1, seem to vary from the other genotypes. The bacterial stem communities are more tightly clustered than those in the fungal stem communities. However, there was still significant community variation with P-values of about 0.04 and 0.01, respectively (Table S8) on genotype. However, the bacterial stem communities are far from the Cherry Wine and other genotypes. For all other organs, genotypes showed high statistically significant differences in both bacterial and fungal community structures (p values, ≤ 1.00E−04), as shown in Table S8.
The LEfSe identifies discriminative fungal and bacterial taxa from the different Hemp genotypes (Fig. 5). When analyzing the LDA effect size (LEfSe, α = 0.05), we focused on discriminative taxa with a p value of < 0.025 due to the smaller sample size. There were no bacterial or fungal taxa that were determined to be discriminative. While there were discriminative bacterial taxa (p < 0.05, none below 0.025), meaning the variation might not be significant. We identified discriminatory bacterial taxa within the roots of all genotypes; all P-values are 0.02 unless noted otherwise. The unknown genotype had 13 identified taxa. Eight of the taxa belonged to the Actinobacteria phylum, those being Lechevalieria, Mycobacterium, Nocardioides, Actinophytocola, Actinoplanes, Kibdelosporangium, and Amycolatopsis. The other taxa are composed of Bacteroidota (genus Niastella, LDA score = 3.55), Proteobacteria (genera Shinella [3.46], SM2D12 [3.09], and Microvirga [3.07]), Bdellovibrionota (genus Bdellovibrio, 3.13), Myxococcota (genus Sandaracinus, 3.08). Genotype Sweet Sensi only had three relevant taxa: two from the phylum Firmicutes, genera Paenibacillus (4.16) and Shimazuella (2.97), and one from the phylum Proteobacteria, genus Pseudomonas (4.78). The Cherry Wine genotype had nine relevant taxa from phyla Proteobacteria, Actinobacteria, Bacteroidota, Chloroflexi, and Myxococcota. The genera of these phyla are Rhizobium (4.26), Methylophilus (3.97), Sphingobacterium (3.72), Nonomuraea (3.40), Cellbivrio (3.28), and Stenotrophomonas (3.26). Lastly, genotype American Victory-1 had four taxa identified family Kineosporiaceae of the phylum Actinobacteria (4.84), order Armatimonadales (3.05), genus Pir4_lineage of phylum Plantomycetota (3.60), and genus Subgroup_10 of family Thermonanaerobaculaceae (3.02).
Cladograms representing specific taxonomic levels determined to be distinct to particular genotypes using LDA effect size (α = 0.05) and Newark tree produced by Qiime2. The legend for the bacteria root cladogram can be found in Supplementary Data Table S7.
However, a significant taxon was present in the other organs (leaf, root, and soil), including fungal taxa identified within these parameters. Soil fungal communities based on genotype had the least variation with only five differential taxa, two belonging to Unknown, another to Sweet Sensi, and one for Cherry Wine. For the Unknown genotype genera, Mariannaea (3.81) and Calvatia (3.57) were represented, and Sweet Sensi Neocosmospora and Thanatephorus were represented (4.91, 3.68, respectively). Lastly, the genotype Cherry Wine had only one discriminatory fungal genus, Gibberella (4.80). Moving up the plant into the root system, nine significant taxa were found overall. The unknown genotype had genera Botryotrichum and Xylaria, and unidentified from order Branch06 of class Sordariomycetes along with family Cerotavasidiaceae (5.06, 4.86, 4.35, and 3.91, respectively). Cherry Wine genotype had genera Neocosmospora (5.20), Athelia (5.16), and Pseudallescheria (4.01) shown as discriminatory taxa. American Victory-1 and Sweet Sensi genotypes had only one distinct taxon found: Candida (4.71) and Alternaria (5.04), respectively. Finally, we will describe the taxa of relevance within the leaves of different genotypes. Sweet Sensi was the most distinct, with four genera of distinction: Zymoseptoria, Meira, Dimorphiseta, and Pseudozyma. Cherry Wine had only one taxon, the family Bionectriaceae, with an LDA score of 3.67. American Victory-1 had two distinct taxa: one of the genera Aspergillus (3.77) and one of the families Chaetomiaceae (3.29). The unknown hemp genotype had no distinct taxa in their leaf fungal communities.
Microbiome diversity based on genotype traits: CBD and fiber-richness
Grouping the genotypes by their production use of either Fiber or CBD, we discovered that only the roots and soil had significantly different bacterial communities (p < 0.002). The fungal communities differed significantly across the soil, root, and stem but not the leaf communities (p < 0.003, 0.05, 0.03, and 0.30, respectively; Fig. 6). Running LEfSe analysis (α = 0.01) on the samples when grouped by production types, some taxa were identified as differentially abundant between the two groups (Fig. 7). Only the rhizosphere had differentially abundant taxa with the strict alpha values (0.01) set due to the small sample size and to reduce false positive findings. Soil bacterial diversity was very similar. The bacterial root diversity was found to have multiple bacterial taxa and was significantly upregulated in Fiber production plants, with the genera Asticcacaulis (3.05) and Shinella (3.37), Saccharimonadales (3.25), Nonomuraea (3.28), Nocardioides (3.57), Niastella (3.35), Mycobacterium (3.95), Blrii41 (3.13), Aeromicrobium (3.77), and Actinoplanes (3.63).
Bray–Curtis distances represented along both Principal Coordinates Analysis (PCoA) 1, 2, and 3 axes. The figure is split vertically by organ and horizontally by bacterial vs fungal diversity. PERMANOVA was run, and all were significantly different except for bacterial and fungal leaf communities, which were not significantly distinct.
Discriminative taxa based on the relative abundance compared to the Hemp plant’s CBD or fiber agricultural production. The larger the confidence interval on the right-hand side shows a higher fluctuation between plants. Orange represents CBD-producing plants, while blue represents Fiber-producing plants. The Figure is again split vertically based on organ and horizontally with bacterial taxa on the left and fungal taxa on the left.
In contrast, fungal diversity had a more significant variation in soil between the plants, with 14 differentially abundant taxa. The genera Neocosmospora (4.77) and Myrothecium (3.33) are of note, with the most significant differentiation in CBD-producing plants. The fungal taxa in the soil near Fiber-producing plants had four distinct genera: Fusicolla (3.88), Cladosporium (3.17), and Triangularia (3.44). The fungal taxa along the root system differed significantly from the genus Pseudallescheria (3.52). The fungal root community for CBD production plants had relevant taxa of genera Alternaria (4.78), Stemphylium (3.17), Candida (4.46), Condenascus (3.64), and Achroiostachys (3.22).
Core-microbiome player of hemp genotypes
Investigating the core microbiome, we found significant variation across CBD vs Fiber genotypes. We found 16 and 11 core microbiome bacterial and fungal species across genotypes (Table S7). The results showed that Sphingomonas, Pseudomonas, and Bacillus were the core microbiome species of fiber genotypes with a highly abundant presence compared to CBD genotypes. Microbacterium and Rhizobium were significantly higher in CBD than fiber. In the case of the fungal microbiome, the Alternaria and Gibberella formed a core microbiome of fiber genotype rather than CBD. Contrarily, Penicillium and Nigrospora were significantly more abundant in CBD than in fiber genotypes (Table S9).
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- Source: https://www.nature.com/articles/s41598-024-79192-7