Microbiome structure of hemp
Microbiome diversity is defined as the number and abundance distribution of specific species of microbes 43,44. Thus, in the current research, we evaluated the microbial diversity and abundance of hemp species rich in CBD (Sweet Sensi and Cherry Wine) and Fiber (American Victory and Unknown). We evaluated both rhizosphere (root and soil) vs. phyllosphere (stem and leaf) in this study. 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 (Table S1, S2, S3). 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 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 recovered from the leaf (53.6%) of CBD 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. The fiber-producing varieties tend to recruit different bacterial and fungal microbiomes compared to 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.
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 8 phyla found in the data, the top 3 phyla were Proteobacteria, Acidobacteria, and Actinobacteria.Exceptf 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).
In the stem and leaves of the plants, we found that Proteobacteria remain the top phylum accounting for almost 97.5% in the Cherry Wine genotype. At the same time, Proteobacteria remains the top phylum for all other genotypes, between 60–98%. Firmicutes (0.5–21%) and Actinobacteria (1–14%) continue to play an important role in the stem, while 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, which were Septoria, Cladosporium, Alternaria, Gibberella, and Moesziomyces. The only genus Moesziomyces belonged to Basidiomycota, whereas most were Ascomycota (Fig. 4).
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, but 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 genotype from the 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 taxa (Fig. 5), both fungal and bacterial, from the different Hemp genotypes. 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 [LDA score = 3.46], SM2D12 [LDA score = 3.09], and Microvirga [LDA score = 3.07]), Bdellovibrionota (genus Bdellovibrio, LDA score = 3.13), Myxococcota (genus Sandaracinus, LDA score = 3.08). Genotype Sweet Sensi only had three relevant taxa two from the phylum Firmicutes, genera Paenibacillus (LDA score = 4.16) and Shimazuella (LDA score = 2.97), and one from the phylum Proteobacteria, genus Pseudomonas (LDA score = 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 (LDA score = 4.26), Methylophilus (LDA score = 3.97), Sphingobacterium (LDA score = 3.72), Nonomuraea (3.40), uncultured_f_Roseiflexaceae (LDA score = 3.40), Cellbivrio (LDA score = 3.28), Stenotrophomonas (LDA score = 3.26), and Blrii41 (LDA score = 3.20). Lastly, genotype American Victory-1 had four taxa identified family Kineosporiaceae of phylum Actinobacteria (LDA score = 4.84), order Armatimonadales (LDA score = 3.05), genus Pir4_lineage of phylum Plantomycetota (LDA score = 3.60), and genus Subgroup_10 of family Thermonanaerobaculaceae (LDA score = 3.02).
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 amount of variation with only five differential taxa, two belonging to Unknown, another two to Sweet Sensi, and one for Cherry Wine. For the Unknown genotype genera, Mariannaea (LDA score = 3.81) and Calvatia (LDA score = 3.57) were represented, and Sweet Sensi Neocosmospora and Thanatephorus were represented (LDA score = 4.91, 3.68 respectively). Lastly, the genotype Cherry Wine had only one discriminatory fungal genus, Gibberella (LDA score = 4.80). Moving up the plant into the root system, there were nine significant taxa found overall. The unknown genotype had genera Botryotrichum, Xylaria, and unidentified from order Branch06 of class Sordariomycetes along with family Cerotavasidiaceae (LDA scores 5.06, 4.86, 4.35, and 3.91, respectively). Cherry Wine genotype had genera Neocosmospora (LDA score = 5.20), Athelia (LDA score = 5.16), and Pseudallescheria (LDA score = 4.01) shown as discriminatory taxa. Both American Victory-1 and Sweet Sensi genotypes had only one distinct taxon found, and those were Candida (LDA score = 4.71) and Alternaria (LDA score = 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 taxa, the family Bionectriaceae with an LDA score of 3.67. American Victory-1 had two distinct taxa one of the genera Aspergillus (LDA score = 3.77) and one of the families Chaetomiaceae (LDA score = 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-value, 0.002). The fungal communities differed significantly across the soil, root, and stem but not the leaf communities, with P-values being 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 regarding only the genus Acidibacter and the order Gamaproteobacteria_Incertae_Sedis having a slightly higher presence around CBD-production type plants (LDA score = 0.04). 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).
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. Where fungal taxa in the soil near Fiber producing plants had four distinct genera Fusicolla (3.88), Cladosporium (3.17), Triangularia (3.44), and an unidentified genus belonging to the family Pezizaceae (3.35). A 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 were forming a core microbiome of fiber genotype than CBD. Contrarily, Penicillium, and Nigrospora were significantly more abundant in CBD than in fiber genotypes (Table S9).