Wild again: recovery of a beneficial Cannabis seed endophyte from low domestication genotypes
https://ror.org/00d7xrm67grid.410413.30000 0001 2294 748XInstitute of Environmental Biotechnology, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria
https://ror.org/00d7xrm67grid.410413.30000 0001 2294 748XInstitute for Signal Processing and Speech Communication, Graz University of Technology, Inffeldgasse 16C/EG, Graz, 8010 Austria
https://ror.org/04d62a771grid.435606.20000 0000 9125 3310Leibniz Institute for Agricultural Engineering and Bioeconomy, Max-Eyth-Allee 100, 1446 Potsdam, Germany
https://ror.org/03bnmw459grid.11348.3f0000 0001 0942 1117Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, Potsdam-Golm, 14476 Germany
School of Biological Sciences, Faculty of Environmental and Life Sciences, Highfield Campus, Southampton, SO17 1BJ UK
Abstract
Background
Beyond carrying the plant embryo, seeds harbour intricate microbial communities whose transmission across successive plant generations can significantly influence the ecological and evolutionary dynamics of plant–microbe symbioses. The process of plant domestication has potential repercussions in genes involved in plant-microbiome interactions. However, the extent to which breeding can impact the seed microbiome is sparsely explored. Cannabis is a high-value crop but sparsely subjected to agricultural innovations established in other crop species during the last century. Here, we conduct a large-scale analysis of the bacterial seed microbiome of Cannabis across different domestication grades and investigate the potential of seed-associated endophytes as plant growth-promoting agents under both controlled and field conditions.
Results
Analysis of Cannabis seed endophyte composition and diversity across 46 plant genotypes revealed 813 different bacterial genera with a predominance of Gammaproteobacteria, Bacilli, Actinobacteria and Alphaproteobacteria but a genotype-specific microbiome. The assessment of domestication and breeding on microbial assembly revealed a higher bacterial diversity in low domestication genotypes (Shannon index, H′: 1.21 vs. 1.05) and a higher homogeneity in bacterial composition caused by line development. Further, a seed bacterial isolate (Bacillus frigoritolerans C1141) associated with low domestication genotypes, and with genes associated with bio-fertilization, bioremediation and phytohormone production, increased plant growth by 42.3% at the time of harvest, under field conditions.
Conclusion
This study addresses critical knowledge gaps related to the assembly of the Cannabis seed-endophytic microbiome. It reveals that Cannabis breeding is linked to alterations of seed microbial communities, which potentially led to the loss of bacteria with functional significance. These results highlight the importance of preserving seed microbiomes in plant breeding to support sustainable plant health and growth enhancement in Cannabis.
Supplementary Information
The online version contains supplementary material available at 10.1186/s40168-024-01951-5.
Background
Plants host microbial communities that are crucial for their growth and vitality [1]. These microbes play multifaceted roles from nutrient mobilization to stress mitigation and enhanced defence against pathogens [2]. Seeds, pivotal in the continuum of plant existence, serve not merely as vessels of plant propagation but as reservoirs of microbial diversity, integral to plant success. Seed-associated microorganisms wield their influence on plants by steering the course of seed germination, preservation, and early development [3]. Furthermore, seed endophytes can be transferred across generations, fostering symbiotic and mutualistic relationships with plants that lay the foundation for robust plant establishment [4–6]. Consequently, seeds emerged as promising sources for the isolation of beneficial microorganisms, as well as targets for microbiome-based breeding strategies [7, 8]. However, a comprehensive understanding of seed microbiome structures and functions remains limited, particularly within the Cannabis genus.
Cannabis, a herbaceous annual plant belonging to the Cannabaceae family, boasts a rich history of domestication alongside significant potential for future utilization [9]. Originating in eastern Asia, Cannabis has transcended borders to become a cosmopolitan crop valued for its versatility in providing fibre, oil, food, and bioactive compounds for medicinal as well as recreational purposes. The human-driven selection across diverse regions has contributed to its extensive diversification [10], resulting in predominantly hybrid genotypes today [11]. Notably, the accumulation of tetrahydrocannabinol (THC), which has psychoactive properties, in the trichomes of female flowers, has posed challenges in resource management and genetic enhancement of Cannabis [12]. Recent trends favour cannabidiol (CBD)-rich genotypes for therapeutic purposes, reflecting a projected global market value of US $75.09 billion by 2029 [13]. However, scaling up Cannabis production brings forth challenges, including heightened susceptibility to fungal pathogens and bacterial infections in greenhouse settings and under field conditions [14]. Additionally, mitigating the environmental impact of cultivation practices is essential to align with climate objectives while meeting production demands [15].
Through the processes of plant domestication and breeding, humans have tailored crops to specific quality traits, reshaping the very essence of plant physiology, morphology, and genetic makeup, which has resulted in a discernible reduction in allelic diversity [16]. These processes might have potential repercussions for genes involved in plant-microbiome interactions and thus affect key traits in plant anatomy, immunity or production of secondary metabolites [17]. For instance, alterations in root morphology and exudate profiles of different plant genotypes can alter the selective recruitment of microorganisms from soil [18–22]. Under domestication, seeds often exhibit the most striking phenotypic alterations, emerging as an optimal domain for exploring those effects on microbial communities [23]. Still, little is known about the intricate nuances of these processes on seed microbial assembly.
Previous studies have outlined the differences in microbial communities of different Cannabis genotypes across plant compartments [24–27]. From those, only a recent study described the variations in bacterial diversity in the Cannabis seed microbiome, however, limited to genotypes with low THC content [28]. Furthermore, the question of whether Cannabis breeding influenced microbial assembly in seeds is left unanswered. Here, we conducted an extensive characterization of the Cannabis bacterial seed microbiome across 46 genotypes with different domestication grades and chemotypes, to cover the high variability of the genus. Seed accessions from early domestication genotypes obtained through natural and artificial selection were included; they are represented by landraces and selected lines. We also included modern genotypes that were obtained through artificial selection; they are represented by accessions classified as cross hybrids and inbred lines. We hypothesized that the heterogeneous genetic background of Cannabis reflects on its seed microbiome. The potential of Cannabis seed endophytes as plant growth-promoting (PGP) and biocontrol agents against different pathogenic fungi has been previously shown [29]. In this work, we tested the potential of Cannabis bacterial endophytes, associated with low domestication genotypes, in promoting growth of Cannabis under controlled and field conditions. We further searched for potential PGP functions in the best-performing bacterium, using whole genome sequencing (WGS). The overall goal of this work is twofold: to comprehensively explore the Cannabis seed microbiome and assess whether this was potentially influenced by domestication, as a strategy for harnessing beneficial seed-endophytic microorganisms that can be used as a sustainable alternative for Cannabis cultivation.
Materials and methods
Preselection of bacterial strains
Bacterial isolation and identification
Bacterial isolates were obtained from surface-sterilized, germinated seeds of Cannabis sativa L. as detailed above and subsequently cultured on nutrient agar (NA II, SIFIN, Berlin, Germany) amended with nystatin (25 μg/mL). For DNA extraction, bacterial cells underwent thermal lysis at 100 °C for 10 min in a buffer (pH 8) containing 10-mM Tris–Cl, 1 mM EDTA, and 1% Triton X-100, followed by rapid cooling to − 20 °C. DNA integrity was verified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The full-length 16S rRNA gene was amplified using primers 27f (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492r (5′-TACGGYTACCTTGTTACGACTT-3′). PCR conditions included an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 61 °C for 30 s, and elongation at 72 °C for 90 s, concluding with a final elongation at 72 °C for 10 min. PCR products were purified with the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and sequenced via Sanger sequencing (LGC Genomics, Berlin, Germany). Sequences were submitted to manual quality filtering with BioEdit 7.7 [53] and analysed using the NCBI BLAST + blastn megablast tool hosted on Galaxy (https://usegalaxy.org/). Only sequences with 100% match to the V4 region of the reference metabarcoding sequences were considered for the identification of the selected bacterial markers.
Bacterial treatments and plant-growing conditions
Selected bacterial strains were cultured individually in Nutrient Broth II (NB II, Difco) at 30 °C overnight until reaching the exponential phase of growth. Cultures were purified through two rounds of centrifugation at 5000 rpm for 10 min, and the resulting bacterial pellets were resuspended in a 0.85% NaCl solution, whereas bacterial concentrations were adjusted to 1 × 108 CFU/mL. Seeds of the Cannabis genotype Fedora 17, an inbred line, were exposed to the different bacterial suspensions for 4 h under agitated conditions at room temperature, followed by germination in sterile CYG germination pouches (MEGA International), each soaked with 15 mL of sterile dH2O. Germinating seeds were kept in dark conditions for 5 days and subsequent 2 days in a greenhouse under a 16-h light cycle at 25 °C. Control seeds were incubated in sterile 0.85% NaCl. The experimental design included three technical replicates per treatment, each comprising 40 seeds. To evaluate the plant growth-promoting effects of the different treatments, data analysis was performed in R v4.2.3 using compare_means from ggpubr [54], applying a nonparametric Wilcoxon rank-sum test with Bonferroni correction for multiple comparisons. The distribution of the variables was assessed using the shapiro.test from stats [41]. For categorical outcomes, the chisq.test with Bonferroni correction for multiple comparisons was employed to analyse the differences between treatments and control.
Field trials
Bacterial treatments and field design
The field trial was conducted at the Experimental Station for Special Cultures (Versuchsstation für Spezialkulturen) in Wies, Austria, within a 187.5 m2 plot divided into three blocks with 1.5 m distance between them. The experimental design included treatment with Bacillus frigoritolerans C1141 (ASV 3055), isolated from the endosphere of Cannabis seeds, and Serratia plymuthica RR2-5–10, isolated from the rhizosphere of oilseed rape cultivated in weakly loamy sand near Rostock, Germany [55]. The latter has been documented for its beneficial effects in various plant systems [56]. Each block consisted of 30 Cannabis plants originated from cuttings of Eletta Campana, a cross hybrid, totalling 90 plants for the experiment. Bacterial stock solutions were prepared from overnight cultures; on the planting day, these cultures were centrifuged, and the cell pellets were resuspended in 0.85% NaCl. These preparations were then diluted to a final concentration of 3 × 107 CFU/mL in the field. The treatment protocol involved immersing the roots of the Cannabis plants in the bacterial suspensions for 5 min before planting, followed by the addition of 50 mL of the suspension to the rhizosphere of each plant. Control plants were treated with the same volume of sterile water. The soil at the Experimental Station for Special Cultures was loamy with a pH of 6.6, 4.4% of organic matter, 18 cmol + /kg calcium, a water retention capacity of 23.7% of its weight and with the following nutrient content: > 200 mg/1000 g of plant-available phosphorus, 526 mg/1000 g of plant-available potassium and 183 mg/100 g of plant-available magnesium. The plot was tilled using Geohobel (Rath) and irrigated before planting. No irrigation, fertilizers, or pesticides were applied during the trial. The concentrations of NO3 and NH4 in the soil were 7 kg/ha and 24 kg/ha, respectively. Recorded climatic conditions from June to September showed average temperatures ranging from 13.7 to 25.9 °C, with total rainfall measuring 155.7 mm. August was the wettest month with 258.4 mm of rain, while September recorded the least precipitation at 49.4 mm. Growth metrics, including plant height and stalk diameter, were collected monthly and final biomass assessments at the study’s conclusion.
Statistical data analysis for plant growth parameters
To evaluate the plant growth-promoting effects, data analysis was performed in R. Differences in growth outcomes between treatments were determined using the compare_means from ggpubr [54], applying a nonparametric Wilcoxon rank-sum test with Bonferroni correction for multiple comparisons. The distribution of the variables was assessed using the shapiro.test from stats [41].
Whole genome sequencing
DNA extraction and PacBio sequencing
DNA from B. frigoritolerans C1141 was extracted from overnight cultures in NB II (OD600 = 2.46) for WGS. The extraction was performed using the MasterPure Complete DNA and RNA Purification Kit (Epicentre, Madison, WI, USA), following the manufacturer’s instructions. Additional steps were incorporated for enhanced lysis efficiency, including supplementation of the lysis buffer with lysozyme and transferring of the samples into a Lysing Matrix E tube containing 1.4-mm ceramic spheres, 0.1-mm silica spheres, and one 4-mm glass sphere for mechanical lysis using a FastPrep-24 instrument (MP Biomedicals) at 6 m/s for 25 s, twice, keeping samples 1 min on ice between repetitions. The concentration of extracted DNA was measured using the Qubit dsDNA HS Assay Kit on a Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the DNA was subjected to PacBio sequencing on a PacBio Sequel II/IIe platform after SMRTbell library construction (Novogene Co., Ltd., UK).
Genome processing and mining
The continuous long-read sequencing (CLR) single reads obtained (n = 370,273) were employed for de novo assembly using Flye v2.9.1 [57], resulting in a closed circular chromosome spanning 5,620,668 bp. To assess genome completeness and contamination levels, the assembled genome was evaluated using CheckM v1.2.2 [58], estimating values of 98.81% and 1.42%, respectively. Taxonomic classification was verified using GTDB-Tk v1.4.1 [59], and gene annotation was performed using DRAM v1.2.2 [60] resulting in the identification of 5518 predicted protein coding sequences. Subsequently, amino acid sequences generated by DRAM were utilized to annotate bacterial PGP functions on PlaBAse v.1.02 employing strict mode blast + hmmer. The assembled genome, along with the associated annotations, was deposited in the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/) under accession number PRJNA1113337.
Results
Diversity of Cannabis seed endophytes changes with domestication grade
Alpha-diversity analysis, as measured by observed ASV richness and Shannon H′ index, was conducted based on a rarefied dataset (Fig. 1) Across the dataset, observed ASV richness ranged from 10 to 177 ASVs, with a median of 34 (IQR = 57–20.25) and Shannon H′ index from 0.14 to 2.58, with a median of 1.18 (IQR = 1.73–0.59) (Table S3). Significant differences in alpha diversity were found between genotypes and domestication grades for both measures (P ≤ 0.001, Kruskal–Wallis). Notably, Shannon H′ index values revealed a higher number of significant differences between genotypes, indicating uneven species abundances as a differentiating factor (Fig. S2). Regarding domestication, inbred lines exhibited the lowest diversity in both measures, followed by cross hybrids, whereas the highest diversity was observed in plants of low domestication grades, i.e. landraces and selected lines (Table S3). Differences in observed ASV richness between chemotypes were also significant (P ≤ 0.001) (Fig. S2). Differences in alpha diversity were still observed within the same provider (Table S4).
Cannabis seed microbiome structure is genotype specific, and line development reduces its dissimilarities
The taxonomic composition of the Cannabis seed microbiome was investigated, revealing 813 different genera in 101 different classes belonging to 38 different phyla. Here, four predominant bacterial classes, Gammaproteobacteria, Bacilli, Actinobacteria and Alphaproteobacteria, collectively represented 99.49% of the reads in the dataset and 65.57% of ASV richness (Fig. 2A). Bacillus was identified as the most abundant genus with 22.09% of the reads, represented by 256 ASVs. This was followed by Pantoea with 54 ASVs covering 21.68% of the respective reads, while Ralstonia accounted for 137 ASVs, representing 21.64% of the reads. Other taxa with relative abundances above 1% were Pseudomonas (10.23%, 222 ASVs), Rhodococcus (5.04%, 65 ASVs), Kosakonia (4.47%, 14 ASVs), Paenibacillus (2.5%, 66 ASVs), Enterobacter (2.3%, 6 ASVs), Methylobacterium-Methylorubrum (1.83%, 44 ASVs), Brevibacillus (1.7%, 30 ASVs), Rathayibacter (1.36%, 51 ASVs) and Sphingomonas (1.07%, 82 ASVs).
To explore the influence of plant genotype and domestication grade on seed microbiome composition, ordination based on the Bray–Curtis dissimilarity matrix was conducted and visualized in a UMAP plot (Fig. 2B). Analysis of variance using adonis2 revealed significant differences in beta diversity among different genotypes (P ≤ 0.001), explaining 53.6% of the variations in bacterial composition. Pairwise comparisons demonstrated significant differences in bacterial community composition between the majority of the genotypes, with exceptions noted for specific pairs (Fig. S3). Furthermore, domestication explained 9.66% of the variations in bacterial composition (P ≤ 0.001). Differences in bacterial composition between chemotypes were also significant and explained 6.14% of the variations in bacterial composition (P ≤ 0.001). Moreover, significant differences in beta diversity among genotypes, domestication grades, and chemotypes within the same provider were observed (Table S4). Samples from genotypes associated with line development, i.e. selected lines and inbred lines, showed more positive silhouette coefficients, indicating a homogenization of their bacterial communities (Fig. 2b).
Bacterial taxa with a relative abundance of at least 5% in the whole dataset were assessed for each genotype, resulting in the identification of 26 genera across five classes (Fig. 2C). Additionally, these were visualized for each sample across genotypes (Fig. S4). While all five classes were ubiquitous, their relative abundances varied. Gammaproteobacteria exhibited the highest diversity, dominating in 31 genotypes, followed by Bacilli in 13. Notably, certain genera were dominant in more plant genotypes, such as Bacillus and Ralstonia in 12 genotypes each and Pantoea and Pseudomonas in 11 and 5 genotypes, respectively. Actinobacteria predominantly occurred in one genotype (C54), primarily due to the genus Rathayibacter. The same was observed in C37 regarding Alphaproteobacteria, largely due to Methylobacterium-Methylorubrum. The class Bacteroidia did not emerge as dominant in any genotype, with its presence mainly observed in C77. The genotype C47 exhibited the highest percentage of reads outside these dominant classes and genera, albeit accounting for a minor proportion of the dataset.
Identification of bacterial markers linked to plant domestication
We found that the seed core microbiome for each of the domestication grades representing ASVs shared between at least 75% of the samples within the group with a detection level of 0.1%. We found a similar number of core species within each domestication grade, however representing different cumulative relative abundances in each grade, whereas selection lines and inbred lines values reached up to 81.65% and 96.88%, respectively, while for landraces and cross hybrids core species represented 67.61% and 51.8%, respectively (Fig. 3A). In contrast, inbred lines are notably dominated by the presence of one ASV, presenting a relative abundance of 82.05%, i.e. Pantoea agglomerans (ASV 3353). Furthermore, 8 out of 17 core species found across grades, such as Pelomonas (ASV 2062), Ralstonia (ASV 2280 and ASV 2328), Burkholderia sp. (ASV 2389), Pseudomonas sp. (ASV 3198), P.agglomerans (ASV 3353), Enhydrobacter (ASV 3464) and Rhodococcus erythropolis (ASV 4544), were ubiquitous to all grades and collectively constitute a large fraction of the entire bacterial community with a cumulative relative abundance of 63.32% in the whole dataset (Fig. S5). Inbred lines and selected lines have shown to harbour more unique core members, including Pseudomonas (ASV 3136), Kosakonia (ASV 3369) and Enterobacter (ASV 3380) in inbred lines and Bacillus (ASV 2970), Dyella (ASV 2505) and BD7-11 (ASV 3820) in selected lines. Landraces only have Sphingomonas (ASV 1215) as the unique core member, while cross hybrids have no unique core members.
The ML-based analysis with XGBoost revealed distinct microbial signatures associated with each domestication grade. ASVs such as Pseudomonas putida (ASV 3151), Bacillus (ASV 3007) and Pseudomonas (ASV 3140) were among the top 10 bacterial genera contributing to classification of landraces (Fig. 3B). Conversely, selection lines exhibited enrichment of taxa such as Rathayibacter (ASV 4090) and Bacillus sp. (ASV 3055). Cross hybrids included taxa such as Bacillus sp. (ASV 2704 and 2970) and Paenibacillus (ASV 4599, 4674, and 4601). Inbred lines had exclusively P. agglomerans (ASV 3353) as their signature, indicating a potential shift in the seed microbiome. These results underscore the potential of using seed microbiome profiles as biomarkers for plant domestication grades and provide insights into potentially beneficial taxa lost during domestication. The abundance and prevalence of the ASVs identified as biomarkers or core members for each domestication grade are presented in Fig. 3C.
Effects of the seed endophyte B. frigoritolerans C1141 on Cannabis growth and fitness
In light of the preceding findings, our investigation delved into the impact of the identified biomarkers from low domestication genotypes on Cannabis growth and fitness under both controlled and field conditions. From the obtained Cannabis seed isolates, a total of five biomarkers, three of landrace genotypes (ASV 3007: Bacillus sp., 3140: Pseudomonas sp., and 3151: P.putida) and two of selected lines (ASV 4090 Rathayibacter sp. and ASV 3055: Bacillus sp.), were identified with a 100% match in the V4 region and selected for further examination (Table S5).
Priming of seeds with the aforementioned bacteria was implemented to assess potential effects on growth under controlled conditions. While all treatments, except for ASV 3140 (Pseudomonas sp.), exhibited enhancements in biomass, root and shoot length in 7-day-old seedlings, these improvements did not reach statistical significance (Fig. S6). Notably, treatment with ASV 3151 (P.putida) significantly increased the germination rate (P ≤ 0.05, χ2) (Fig. 4A), and the treatment with ASV 3055 (Bacillus sp.) emerged as the sole intervention to significantly improve secondary root formation (P ≤ 0.05, χ2) and mitigate the incidence of dead seedlings with dry stems. The latter was further identified as B. frigotolerans, assigned as strain C1141 and selected for further investigation under field conditions.
During field trials, the effects of B. frigoritolerans C1141 on Cannabis plants were compared with those of the patented plant-beneficial bacterium S.plymuthica RR2-5–10, as well as with untreated Cannabis plants. Results showed discernible differences in plant fresh weight upon harvest between the treatment groups and the control, with average yields 3 × higher in plants treated with B. frigoritolerans C1141 and 2 × higher in plants treated with S.plymuthica RR2-5–10 (Fig. 4B). Furthermore, treatment groups exhibited significant differences in plant height over the course of the trial, with marked improvements observed in plants treated with B. frigoritolerans C1141 from the second timepoint on and 43.3% higher at harvest, in contrast to those treated with S.plymuthica RR2-5–10, where an improvement in growth was only evident at the final timepoint (11.5%) (Fig. 4C). Remarkably, plants treated with B. frigoritolerans C1141 demonstrated a significant increase in stalk diameter as early as the first month (timepoint 1), a trend that persisted throughout the duration of the trial, reaching improvements of 96% at the time of harvest, which more than doubles the effects of S.plymuthica RR2-5–10 (42%) (Fig. 4D). These results provided a reliable confirmation of the benefits and adaptability of the seed microbiota when it comes to target approaches for the improvement of plant growth and fitness.
Finally, we conducted a comprehensive analysis of strain C1141’s PGP functions using PLaBase. This approach offers a thorough characterization through genetic analysis, offering detailed insights into the multifaceted mechanisms underlying the potential of strain C1141 as an effective agent for promoting plant growth. A diverse array of 1572 distinct protein coding genes or gene clusters associated with various PGP functions was found and poised positively to impact plant growth, both directly (n = 591) and indirectly (n = 1315) (Fig. 5). Among the notable findings were numerous genes facilitating plant colonization (n = 678), particularly through
the utilization of plant-derived substrates (n = 554) and mechanisms of motility or chemotaxis (n = 60), which are instrumental for seed-endophytic colonization via internal pathways. Additionally, a multitude of genes linked to phytohormone production (n = 254) were identified, with a notable abundance of genes involved in plant vitamin production (n = 132), volatile metabolism (n = 46), and germination stimulation (n = 40). A substantial number of genes associated with bio-fertilization (n = 263) were uncovered, primarily involved in phosphate (n = 120) and potassium (n = 93) solubilization, as well as iron (n = 62) and nitrogen (n = 55) acquisition. Moreover, our analysis revealed genes implicated in immune response stimulation (n = 51), particularly those involved in inducing systemic resistance (ISR) (n = 41), and in stress management or biocontrol (n = 445), aimed at mitigating both biotic (n = 111) and abiotic (n = 306) stress factors. Genes associated with competitive exclusion (n = 490), indicative of bacterial fitness (n = 162), and with mechanisms of quorum sensing or biofilm formation (n = 98) were identified, underscoring their importance in facilitating persistence within the plant ecosystem. Detailed descriptions of the top 25 gene features and associated genes are provided in Table S6.
Discussion
In this work, we provide new insights to support harnessing the Cannabis seed microbiome for optimizing crop performance and sustainability in agricultural systems. The plant microbiome plays a pivotal role in shaping plant health and productivity. The process of domestication may have inadvertently disrupted the microbial equilibrium and symbiosis within currently produced plants. This could also include alterations in the microbiome composition and function that are fundamental for optimal growth and resilience against environmental stresses. Given the significant importance of seeds in ensuring the continuity of beneficial microbes, understanding the effects of domestication on the seed microbiome, particularly in economically significant crops like Cannabis, is paramount.
Here, we found evidence that the plant genotype is determinant in explaining the variation in the Cannabis seed bacterial diversity and composition. Although previous research has elucidated the pivotal role of the plant genotype in shaping the assembly of the seed microbiome [61], in Cannabis, it is a novel insight covering a highly heterogeneous genetic background. In analogy to other plant species, the diversity of Cannabis seed microbiome is lower than in other plant compartments [24, 25, 27]. The low moisture content and restricted nutrient availability inherent to seeds create an inhospitable environment for microbial endurance and proliferation [62]. Therefore, seeds are considered a bottleneck in the continuity of the plant microbiome. When compared to other plant species, Cannabis seeds harbour bacterial communities with low to intermediate diversity [63]. Here, differences in evenness of the bacterial communities were more determinant to distinguishing between genotypes than species richness. We have also found well-marked differences between the seed-endophytic community compositions of different Cannabis genotypes. These observations are in accordance with the strong effect of the plant genotype found in other above-ground compartments of Cannabis [24–27] and are supported by a smaller scale study with 16 genotypes [28]. Similar observations were being made in the seed-endophytic communities of other plant species [64–66]. The plant genotype generally shows a higher influence on these microbial communities than geographic location [67–70]. Although in the present study differences among plant genotypes from the same provider revealed selection by the host, pre- and postharvest variations, such as location, environmental fluctuations or even conditions and duration of storage, can still remain influential [71–73]. A detailed untangling of the interplay between host genetics and environmental effects on Cannabis seed microbiome assembly will require targeted experiments.
Domestication was also identified as a significant factor in shaping the seed-endophytic communities of Cannabis. Domestication, and breeding, wielded adeptly, can manipulate the genetic makeup of plants, leading to drastic changes in the seed microbiome, which has also been observed in other plants. Interestingly, the seed community structures differed between domestication grades, and genotypes originating from line development exhibited a higher clustering in comparison to landraces and cross hybrids. Hybridization between different genotypes can introduce novel genetic elements into the plant genome, potentially altering the interactions between the plant and its associated microbiota [74]. On the other hand, inbreeding involves repeated crosses within a limited gene pool, which can lead to the fixation of certain alleles and the loss of others through genetic drift [75]. Similarly, line development involves selecting and propagating individuals with specific traits over multiple generations, often within a narrow genetic background. While these breeding approaches aim to stabilize desired traits, they may inadvertently reduce genetic diversity and the potential for introducing novel genetic elements into the plant genome that could contribute to microbial diversity and interactions. In the present study, a significantly higher bacterial diversity was observed in low domestication grade genotypes (landraces and selected lines) in comparison to high domestication grade (cross hybrids and inbred lines), whereas selected lines showed the highest diversity. Studies have drawn diverging outcomes in this regard [76, 77], which reveal that the effects of domestication on the seed microbiome diversity are dependent on plant species.
In our study, differences between the bacterial composition of low and high THC genotypes were found. In Cannabis, breeding initiatives often coincide with the manipulation of cannabinoid ratios and potency and terpenoid profiles [78]. However, the impact of the interplay between breeding and secondary metabolites on the seed microbiota remains largely unexplored [79]. In a recent study, THC and THC acid were found to exhibit genotoxic effects towards certain bacteria, while other cannabinoids can show cytotoxic or oxidative effects [80]. It is likely that the accumulation of secondary metabolites within the trichomes of Cannabis flowers can act as a deterrent to pests and pathogens and thereby influence the microbial environment surrounding the developing seed.
Here, we used a broad range of Cannabis genotypes to comprehensively explore the bacterial community in Cannabis seed and the potential effects of different chemotypes. From the 101 different bacterial classes found, five — Gammaproteobacteria, Bacilli, Actinobacteria, Alphaproteobacteriaand Bacteroidia — were ubiquitous to all genotypes. Endophytic bacteria of the same phylogeny are commonly found in the seed endosphere of other plants [63]. In Cannabis, these bacteria have been previously reported in other plant compartments. For instance, bacteria belonging to Firmicutes and Proteobacteria — mostly Bacillus and Pseudomonas — were predominantly detected in the flower and leaf endospheres, while Sphingomonas and Methylobacterium have been previously found on their surfaces and Actinobacteria on the root endosphere [24, 26]. This can allude to recruitment from plant to seed via the internal and floral pathways [81]. Furthermore, these classes do not differ from the ones reported in hemp seeds [28]. However, genera like Flavobacterium, Acidovorax, Herminiimonas, Chryseobacterium and Massilia, therein described as most abundant, compose members of the rare taxa in our study, with relative abundances similar or less than 0.01%. Moreover, neither Flavobacterium nor Herminiimonas are highly prevalent among the analysed genotypes in our study. Such differences could have arisen from the harsh sterilization method applied by the authors. A previous study focusing on the surface sterilization effects on endophytes in tea leaves and stems indicated that Bacillus has the highest susceptibility to high concentrations of NaClO [82]. The extent to which these treatments can affect certain types of taxa and yield false negatives can be dependent on their exact localization inside the seed tissues.
The spanning diversity of Cannabis genotypes tested, covering great part of its versatility and the polymorphic amplitude, allowed to refine the identification of shared ASVs. We found that both selected and inbred lines harboured more unique core members in comparison to the other domestication grades. Herein, ASVs shared within the same domestication grade and can point to potentially important M gene-bacteria associations that were affected by the domestication process. M genes, short for microbiome genes, were recently introduced to classify plant genes that are linked to the microbiome [83]. In addition, generally shared ASVs, ubiquitous to all domestication grades, can provide evidence of evolutionary conservation independently of domestication and chemotype. Core genera like Pantoea, Pseudomonas, Ralstonia and Burkholderia seem to be widespread across seeds of different plant species, which may point to important physiological functions in spermatophytes [63]. From the core microbiome of low to high domestication grades, we observed an enrichment of members of the order Enterobacteriales, i.e. Kosakonia and Enterobacter and Pantoea, in the core microbiome of inbred lines and a reduction/depletion of Bacillales, i.e. Staphylococcus and Bacillus. Notably, a large proportion of P. agglomerans (ASV 3353) was found in the core of inbred lines. P. agglomerans has known plant growth promoting [84, 85], as well as antagonistic properties towards phytopathogens [86], and can be vertically transmitted across plant generations [87]. Previous research has consistently detected a higher abundance of Bacteroidetes in the root-associated microbiome of wild relatives, while an enrichment of Actinobacteria and Proteobacteria was observed with modern crops [88, 89]. These observations might convey the influence of soil management practices and are not fully translated to the microbiome of seeds. In seeds, the influence of domestication on the microbial communities has been reported to be dependent on the resulting plant phenotype [90], and an enrichment in Pantoea has been associated with intensive breeding in other species [76, 77, 91].
While it is not clear what consequences this enrichment might have in terms of plant fitness, it would be interesting to address which M genes might be responsible for it.
Based on the diversity assessments performed in this study, we decided to search for particular ASVs that may support growth or fitness of Cannabis plants but were lost during the domestication process, as suggested by Raaijmakers and Kiers [92]. Hence, we consider shifts in the seed microbiome not only a reflection of the domestication state of the plant but also responsible for plant fitness. The present study used rather short 16S rRNA gene fragments to confirm sequence matches between amplicon data and sequences obtained from isolates. Future approaches would profit from full-length 16S rRNA sequencing of amplicons from seed material for more precise identifications.
The field trial results conducted in this study confirmed the capability of the selected isolate B. frigoritolerans C1141 to promote growth in Cannabis cuttings from a cross hybrid. Most notably, this bacterium outperformed the beneficial effects of the already established plant beneficial S. plymuthica RR2-5–10, isolated from the rhizosphere of oilseed rape. These results not only supported seeds, as a useful source of beneficial microorganisms, but also that the modulation of the seed microbiome through personalized interventions may help to restore plant homeostasis and improve plant health. Moreover, these results also apprise to the importance of under-domesticated genotypes like locally adapted landraces, which can provide a repository to reintroduce microbial diversity into domesticated populations. This is especially true in Cannabis whose natural seed microbial transmission got interrupted by phytosanitary requirements or vegetative propagation practices [93]. Furthermore, the comprehensive characterization of B. frigoritolerans C1141 genomic features provided important insights in our understanding of its functional capabilities. This strain harboured a considerable number of genes and gene clusters related to plant colonization, phytohormone production, bio-fertilization, immune response stimulation, stress control and competitive exclusion. These genetic features can explain not only its ability to successfully colonize Cannabis plants and eventually the seeds but also the observed beneficial outcomes in Cannabis.
Conclusion
Our study provides new insights related to the impact of plant genotype, domestication and breeding on endophytic bacterial communities in Cannabis seeds. Cannabis genotypes shared a fraction of predominant bacteria but presented significant differences in terms of diversity and composition. Our data showed further microbial shifts related to domestication and breeding in terms of composition, diversity and abundance. Within the frame of the rewilding hypothesis, we were able to promote Cannabis fitness when reinstating a bacterial seed endophyte associated with low domestication. This work highlights the potential of leveraging seed-associated microorganisms to enhance plant growth and resilience and the importance of considering microbiome-assisted crop improvement or fine-tuning seed endophyte composition via M gene breeding for sustainable Cannabis production by reducing the need for chemical inputs.
Supplementary Information
Abbreviations
- ASVs
- Amplicon sequencing variants
- AUC
- Area under the receiver operating curve
- CBD
- Cannabidiol
- CLR
- Continuous long-read sequencing
- CSS
- Cumulative sum scaling
- IQR
- Interquartile range
- ML
- Machine learning
- NaClO
- Sodium hypochlorite
- NA II
- Nutrient agar II
- NB II
- Nutrient broth II
- OD600
- Optical density at a wavelength of 600 nm
- PGP
- Plant growth promoting
- PNA
- Peptide nucleic acid
- SHAP
- SHapley Additive exPlanations
- THC
- Tetrahydrocannabinol
- WGS
- Whole genome sequencing
Acknowledgements
The authors would like to thank the staff of the Experimental Station for Special Cultures (Versuchsstation für Spezialkulturen) in Wies, Austria, and particularly Doris Lengauer, for their extensive support during the field trials and for providing the data on soil properties. Furthermore, we would like to thank Katherine Cuadros-Patiño from the Institute of Environmental Biotechnology (TU Graz) for conducting preliminary laboratory trials, Monika Schneider-Trampitsch for helping with the preparation of the field trials, and Matthias Schweitzer, Kristina Michl, Nina Bziuk, Jasper Schierstaedt, Anna Mitterrutzner, Mathias Eisenhut and Rok Pekušek for helping during the field trials. This publication was supported by TU Graz Open Access Publishing Fund.
Funding
Open access funding provided by Graz University of Technology. This study received funding from Austrian Research Promotion Agency (FFG) in the frame of the CannaBiom project (no. 883706) awarded to TC.
Data availability
The assembled Bacillus frigotolerans C1141 genome, along with the associated annotations, was deposited in the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/) under accession number PRJNA1113337. The 16S rRNA gene amplicon dataset was deposited in the European Nucleotide Archive (ENA; https://www.ebi.ac.uk/ena) under the accession number PRJEB64469. The bioinformatic and statistical analysis pipeline and files for reproducibility are available in the GitHub repository, https://github.com/cbclobato/wild-again.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.