Microbiome of Field Grown Hemp Reveals Potential Microbial Interactions With Root and Rhizosphere Soil

Abstract

Hemp (Cannabis sativa L.) is a crop bred and grown for the production of fiber, grain, and floral extracts that contribute to health and wellness. Hemp plants interact with a myriad of microbiota inhabiting the phyllosphere, endosphere, rhizoplane, and rhizosphere. These microbes offer many ecological services, particularly those of below ground biotopes which are involved in nutrient cycling, uptake, and alleviating biotic and abiotic stress. The microbiota communities of the hemp rhizosphere in the field are not well documented. To discover core microbiota associated with field grown hemp, we cultivated single C. sativa cultivar, "TJ's CBD," in six different fields in New York and sampled hemp roots and their rhizospheric soil. We used Illumina MiSeq amplicon sequencing targeting 16S ribosomal DNA of bacteria and ITS of fungi to study microbial community structure of hemp roots and rhizospheres. We found that Planctobacteria and Ascomycota dominated the taxonomic composition of hemp associated microbial community. We identified potential core microbiota in each community (bacteria: eight bacterial amplicon sequence variant - ASV, identified as Gimesia maris, Pirellula sp. Lacipirellula limnantheis, Gemmata sp. and unclassified Planctobacteria; fungi: three ASVs identified as Fusarium oxysporum, Gibellulopsis piscis, and Mortierella minutissima). We found 14 ASVs as hub taxa [eight bacterial ASVs (BASV) in the root, and four bacterial and two fungal ASVs in the rhizosphere soil], and 10 BASV connected the root and rhizosphere soil microbiota to form an extended microbial communication in hemp. The only hub taxa detected in both the root and rhizosphere soil microbiota was ASV37 (Caulifigura coniformis), a bacterial taxon. The core microbiota and Network hub taxa can be studied further for biocontrol activities and functional investigations in the formulation of hemp bioinoculants. This study documented the microbial diversity and community structure of hemp grown in six fields, which could contribute toward the development of bioinoculants for hemp that could be used in organic farming.

Keywords: Cannabis sativa; bacterial communities; fungal communities; microbial ecology; microbiome; network analysis; rhizosphere.

FIGURE 1

Schematic illustration showing the experimental workflow of a clonal cultivar of hemp grown in six fields distributed at the State of New York (A). MiSeq sequencing and the DADA2 pipeline were used to determine community composition, and core microbes of roots and rhizosphere soils. Network analysis was performed to provide insight into the core microbial taxa and hub microbes. (B) Hypothetical core microbes of roots and rhizosphere soils selected from bulk soil microbiome. Rhizosphere soil is show by blue line surrounding roots.

FIGURE 2

Microbial species diversity and evenness in different hemp fields. The analysis of alpha diversity indices (Shannon and Simpson) and Pielou’s evenness index of the bacterial and fungal communities: (A) bacterial microbiota; (B) fungal microbiota. Indices are shown according to fields. Field name: BGL, Bluegrass Lane; HVL, Hudson Valley Lab; LIH, Long Island Horticultural Research and Extension Center; MC, McCarthy CBD Trial; MSC, McCarthy Stress Trial; and WOF, Wegman’s Organic Farm. Three replicates were used for the analysis of species diversity and evenness.

FIGURE 3

Structure of the community. Principal coordinates analysis (PCoA) showing the community compositions assignments of (A) bacterial 16S rDNA and (B) fungal ITS genes. The variance in the ordinations’ axes 1 and 2 is displayed in parenthesis. Samples from the rhizosphere soil and root are shown by circular and triangle shapes, respectively. Three replicates were used for PCoA analysis. Each color represents filed name: BGL, Bluegrass Lane; HVL, Hudson Valley Lab; LIH, Long Island Horticultural Research and Extension Center; MC, McCarthy CBD Trial; MSC, McCarthy Stress Trial; and WOF, Wegman’s Organic Farm.

FIGURE 4

Taxonomic hierarchy abundance at the order level for bacterial communities. (A) Taxonomic representation at order level in root; (B) relative abundance of top 10 bacterial orders in root. (C) Taxonomic representation at order level in rhizosphere soil (D) relative abundance of top 10 bacterial orders in rhizosphere soil. Three replicates were used for this study.

FIGURE 5

Taxonomic hierarchy abundance at the order level for fungal communities. (A) Taxonomic representation at order level in root; (B) relative abundance of top 10 fungal orders in root. (C) Taxonomic representation at order level in rhizosphere soil (D) relative abundance of top 10 fungal orders in rhizosphere soil. Three replicates were used for this analysis.

FIGURE 6

Indicator species of hemp microbiome. The most representative species of hemp microbiome were identified through indicator species analysis in six different field locations. Bacterial indicator species in root (A) and in rhizosphere soil (B); and fungal indicator species in root (C); and in rhizosphere soil (D). Fungal indicator species in root identified only in WOF filed. Three replicates were used for analyzing indicator species in each field.

FIGURE 7

Interkingdom network cooccurrence in hemp microbiome. The microbial network cooccurrence includes three fully independent replicates. Betweenness centrality and node degree of ASVs and their inter-connection identified eight hub taxa in the root (A). Betweenness centrality and node degree of ASVs and their inter-connection identified six hub taxa in rhizosphere soil (B). Here soil refers to rhizosphere soil. Tables next to each figure identifies microbiota corresponding to the hub ASVs.

FIGURE 8

Network hub taxa identified across six hemp fields in New York. Interkingdom interactions of hub taxa in the root and rhizosphere soil was analyzed using three replicates. The co-association of each hub taxa and its co-associated ASVs is denoted as a Module and ASV connecting between Modules considered as connector taxa. Hub taxa of the module and connector taxa combinedly considered as Network hub taxa. Here soil refers to rhizosphere soil.