Analyzing the Hemp Microbiota
Isolation and Characterization of Endophytic Bacteria Associated to Cannabis sativa cv. Luma as a First Step towards Bioinput Development
DOI:
https://doi.org/10.31285/AGRO.30.1834Keywords:
cannabis, microbiota, health, plant growth promotion, plant protectionAbstract
Plants are naturally associated with microorganisms' communities (microbiotas), which have a significant influence on the plant phenotype, extending its genetic potential and conferring essential traits for growth, resilience, and health. Within these microbiotas, bacterial endophytes are of particular relevance due to their widely distributed plant growth-promoting (PGP) activities and their capacity to synthesize secondary metabolites. These features make them promising candidates for biotechnological applications, particularly the development of bioinputs (biofertilizers, biostimulants, and biopesticides), aimed at reducing dependence on chemical inputs in agriculture. Current hemp (Cannabis sativa) cultivation practices rely heavily on chemical inputs, generating economic, environmental, and social challenges. This study reports the construction and characterization of a collection of endophytic bacteria associated with healthy plants and seeds of the hemp cultivar Luma, a crop cultivated in Uruguay primarily for fiber production. A total of 236 bacterial isolates were obtained from surface-disinfected seeds, seedlings, roots, and aerial tissues, confirming their status as putative endophytes. Of these, 66% of the isolates exhibited at least one of the 5 PGP traits tested, including nitrogen fixation, mineral solubilization, and phytohormone production. Genera with recognized PGP potential, such as Bacillus and Pseudomonas, were particularly abundant in seeds and roots, respectively. Notably, this is the first report describing the association of Mycolicibacterium and Aminobacter strains with cannabis plants. This work represents a first step toward developing bioinputs tailored to the Uruguayan cannabis industry and highlights the broader promise of endophytes for sustainable agricultural practices.
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Ahmad, W., Coffman, L., Ray, R. L., Balan, V., Weerasooriya, A., & Khan, A. L. (2024). Microbiome diversity and variations in industrial hemp genotypes. Scientific Reports, 14(1), Article 29560. https://doi.org/10.1038/s41598-024-79192-7
Alcántara, I., Somma, A., Chalar, G., Fabre, A., Segura, A., Achkar, M., Arocena, R., Aubriot, L., Baladán, C., Barrios, M., Bonilla, S., Burwood, M., Calliari, D. L., Calvo, C., Capurro, L., Carballo, C., Céspedes-Payret, C., Conde, D., Corrales, N., Cremella, B., … García-Rodríguez, F. (2022). A reply to "Relevant factors in the eutrophication of the Uruguay River and the Río Negro". The Science of the Total Environment, 818, Article 151854. https://doi.org/10.1016/j.scitotenv.2021.151854
Avakyan, Z. A., Pivovarova, T. A., & Karavaiko, G. I. (1986). Properties of a new species, Bacillus mucilaginosus. Mikrobiologiya, 55, 477–482.
Balbuena, S., Castelli, L., Zunino, P., & Antúnez, K. (2023). Effect of chronic exposure to sublethal doses of imidacloprid and nosema ceranae on immunity, gut microbiota, and survival of Africanized honey bees. Microbial Ecology, 85(4), 1485-1497. https://doi.org/10.1007/s00248-022-02014-8
Baldani, J. I., Reis, V. M., Videira, S. S., Boddey, L. H., & Baldani, V. L. D. (2014). The art of isolating nitrogen-fixing bacteria from non-leguminous plants using N-free semi-solid media: A practical guide for microbiologists. Plant and Soil, 384, 413-431. https://doi.org/10.1007/s11104-014-2186-6
Balthazar, C., Joly, D. L., & Filion, M. (2022). Exploiting beneficial Pseudomonas spp. for cannabis production. Frontiers in Microbiology, 12, Article 833172. https://doi.org/10.3389/fmicb.2021.833172
Barnett, S. E., Cala, A. R., Hansen, J. L., Crawford, J., Viands, D. R., Smart, L. B., Smart, C. D., & Buckley, D. H. (2020). Evaluating the microbiome of hemp. Phytobiomes Journal, 4(4), 351-363. https://doi.org/10.1094/PBIOMES-06-20-0046-R
Battistoni, F., Fernández Scavino, A., Ferrando, L., Montañez, A., Pezanni, F., Taulé, C., & Vaz-Jauri, P. (2023). Endophytic and rhizospheric microbial communities associated with native and introduced cultivated plant species in Uruguay as sources for plant growth promotion bioinoculant development. Environmental Sustainability, 6, 135–147. https://doi.org/10.1007/s42398-023-00277-6
Berg, G., Grube, M., Schloter, M., & Smalla, K. (2014). The plant microbiome and its importance for plant and human health. Frontiers in Microbiology, 5, Article 491. https://doi.org/10.3389/fmicb.2014.00491
Beringer, J. E. (1974). R factor transfer in Rhizobium leguminosarum. Journal of General Microbiology, 84(1), 188-198. https://doi.org/10.1099/00221287-84-1-188
Castelli, L., Balbuena, S., Branchiccela, B., Zunino, P., Liberti, J., Engel, P., & Antúnez, K. (2021). Impact of chronic exposure to sublethal doses of glyphosate on honey bee immunity, gut microbiota and infection by pathogens. Microorganisms, 9(4), Article 845. https://doi.org/10.3390/microorganisms9040845
Castelli, L., Branchiccela, B., Zunino, P., & Antúnez, K. (2023). Insights into the effects of sublethal doses of pesticides glufosinate-ammonium and sulfoxaflor on honey bee health. The Science of the Total Environment, 868, Article 161331. https://doi.org/10.1016/j.scitotenv.2022.161331
Cavalcante, V. A., & Dobereiner, J. (1988). A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane. Plant and Soil, 108, 23-31. https://doi.org/10.1007/BF02370096
Comeau, D., Balthazar, C., Novinscak, A., Bouhamdani, N., Joly, D. L., & Filion, M. (2021). Interactions between Bacillus Spp., Pseudomonas Spp. and Cannabis sativa promote plant growth. Frontiers in Microbiology, 12, Article 715758. https://doi.org/10.3389/fmicb.2021.715758
Compant, S., Clément, C., & Sessitsch, A. (2010). Plant growth-promoting bacteria in the rhizo- and endosphere of plants: Their role, colonization, mechanisms involved and prospects for utilization. Soil Biology and Biochemistry, 42(5), 669-678. https://doi.org/10.1016/j.soilbio.2009.11.024
Compant, S., Samad, A., Faist, H., & Sessitsch, A. (2019). A review on the plant microbiome: Ecology, functions, and emerging trends in microbial application. Journal of Advanced Research, 19, 29-37. https://doi.org/10.1016/j.jare.2019.03.004
Davies, J., Hawkins, S., Winters, A., & Farrar, K. (2024). Bacterial endophytic community composition varies by hemp cultivar in commercially sourced seed. Environmental Microbiology Reports, 16(2), Article e13259. https://doi.org/10.1111/1758-2229.13259
de los Santos, M. C., Taulé, C., Mareque, C., Beracochea, M., & Battistoni, F. (2016). Identification and characterization of the part of the bacterial community associated with field-grown tall fescue (Festuca arundinacea) cv. SFRO Don Tomás in Uruguay. Annals of Microbiology, 66, 329-342. https://doi.org/10.1007/s13213-015-1113-2
Del Barrio-Duque, A., Ley, J., Samad, A., Antonielli, L., Sessitsch, A., & Compant, S. (2019). Beneficial endophytic bacteria-Serendipita indica interaction for crop enhancement and resistance to phytopathogens. Frontiers in Microbiology, 10, Article 2888. https://doi.org/10.3389/fmicb.2019.02888
Dini-Andreote, F. (2020). Endophytes: The second layer of plant defense. Trends in Plant Science, 25(4), 319-322. https://doi.org/10.1016/j.tplants.2020.01.007
Escobar Rodríguez, C., Antonielli, L., Mitter, B., Trognitz, F., & Sessitsch, A. (2020). Heritability and functional importance of the Setaria viridis bacterial seed microbiome. Phytobiomes Journal, 4(1), 40-52. https://doi.org/10.1094/PBIOMES-04-19-0023-R
Etalo, D. W., Jeon, J. S., & Raaijmakers, J. M. (2018). Modulation of plant chemistry by beneficial root microbiota. Natural Product Reports, 35(5), 398-409. https://doi.org/10.1039/c7np00057j
Ferrari, E., Taulé, C., Mareque, C., Gonzalez, A., Dourron, J., & Battistoni, F. (2023). Unravelling the plant growth promotion potential of the bacterial endophytic microbiota associated with canola (Brassica napus) plants. Environmental Sustainability, 6, 403-413. https://doi.org/10.1007/s42398-023-00289-2
Goyenola, G., Kruk, C., Mazzeo, N., Nario, A., Perdomo, C., Piccini, C., & Meerhoff, M. (2021). Producción, nutrientes, eutrofización y cianobacterias en Uruguay: Armando el rompecabezas. INNOTEC, (22), Article e558. https://doi.org/10.26461/22.02
Gupta, R. S., Lo, B., & Son, J. (2018). Phylogenomics and comparative genomic studies robustly support division of the genus Mycobacterium into an emended genus Mycobacterium and four novel genera. Frontiers in Microbiology, 9, Article 67. https://doi.org/10.3389/fmicb.2018.00067
Guttman, D. S., McHardy, A. C., & Schulze-Lefert, P. (2014). Microbial genome-enabled insights into plant-microorganism interactions. Nature Reviews: Genetics, 15(12), 797-813. https://doi.org/10.1038/nrg3748
Hardoim, P. R., van Overbeek, L. S., Berg, G., Pirttilä, A. M., Compant, S., Campisano, A., Döring, M., & Sessitsch, A. (2015). The hidden world within plants: Ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiology and Molecular Biology Reviews, 79(3), 293-320. https://doi.org/10.1128/MMBR.00050-14
Holmes, J. E., Sanghera, H., & Punja, Z. K. (2023). Crown gall development on cannabis (Cannabis sativa L., marijuana) plants caused by Agrobacterium tumefaciens species-complex. Canadian Journal of Plant Pathology, 45(5-6), 433-445. https://doi.org/10.1080/07060661.2023.2224776
Johnston-Monje, D., Vergara, L. I., Lopez-Mejia, J., & White, J. F. (2023). Plant microbiomes as contributors to agricultural terroir. Frontiers in Agronomy, 5, Article 1216520. https://doi.org/10.3389/fagro.2023.1216520
Kim, S.-J., Lee, C.-M., Han, B.-R., Kim, M.-Y., Yeo, Y.-S., Yoon, S.-H., Koo, B.-S., & Jun, H.-K. (2008). Characterization of a gene encoding cellulase from uncultured soil bacteria. FEMS Microbiology Letters, 282(1), 44-51. https://doi.org/10.1111/j.1574-6968.2008.01097.x
Kruk, C., Segura, A., Piñeiro, G., Baldassini, P., Pérez-Becoña, L., García-Rodríguez, F., Perera, G., & Piccini, C. (2023). Rise of toxic cyanobacterial blooms is promoted by agricultural intensification in the basin of a large subtropical river of South America. Global Change Biology, 29(7), 1774-1790. https://doi.org/10.1111/gcb.16587
Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Molecular Biology and Evolution, 35(6), 1547-1549. https://doi.org/10.1093/molbev/msy096
Lobato, C., de Freitas, J. M., Habich, D., Kögl, I., Berg, G., & Cernava, T. (2024). Wild again: Recovery of a beneficial Cannabis seed endophyte from low domestication genotypes. Microbiome, 12(1), Article 239. https://doi.org/10.1186/s40168-024-01951-5
Lyu, D., Backer, R., Berrué, F., Martinez-Farina, C., Hui, J. P. M., & Smith, D. L. (2023). Plant Growth-Promoting Rhizobacteria (PGPR) with microbial growth broth improve biomass and secondary metabolite accumulation of Cannabis sativa L. Journal of Agricultural and Food Chemistry, 71(19), 7268-7277. https://doi.org/10.1021/acs.jafc.2c06961
Lyu, D., Zajonc, J., Pagé, A., Tanney, C. A. S., Shah, A., Monjezi, N., Msimbira, L. A., Antar, M., Nazari, M., Backer, R., & Smith, D. L. (2021). Plant holobiont theory: The phytomicrobiome plays a central role in evolution and success. Microorganisms, 9(4), Article 675. https://doi.org/10.3390/microorganisms9040675
Mareque, C., & Battistoni, F. (2025). Endophytic interaction of the plant growth-promoting strain Pantoea dispersa UYSB45 and sweet sorghum (Sorghum bicolor) plants. Plant and Soil, 515, 407-422. https://doi.org/10.1007/s11104-025-07598-x
Mareque, C., Taulé, C., Beracochea, M., & Battistoni, F. (2015). Isolation, characterization and plant growth promotion effects of putative bacterial endophytes associated with sweet sorghum (Sorghum bicolor (L.) Moench). Annals of Microbiology, 65, 1057-1067. https://doi.org/10.1007/s13213-014-0951-7
Martínez-Rosales, C., & Castro-Sowinski, S. (2011). Antarctic bacterial isolates that produce cold-active extracellular proteases at low temperature but are active and stable at high temperature. Polar Research, 30, Article 7123. https://doi.org/10.3402/polar.v30i0.7123
Maynaud, G., Willems, A., Soussou, S., Vidal, C., Mauré, L., Moulin, L., Cleyet-Marel, J. C., & Brunel, B. (2012). Molecular and phenotypic characterization of strains nodulating Anthyllis vulneraria in mine tailings, and proposal of Aminobacter anthyllidis sp. nov., the first definition of Aminobacter as legume-nodulating bacteria. Systematic and Applied Microbiology, 35(2), 65-72. https://doi.org/10.1016/j.syapm.2011.11.002
Mirza, B. S., & Rodrigues, J. L. (2012). Development of a direct isolation procedure for free-living diazotrophs under controlled hypoxic conditions. Applied and Environmental Microbiology, 78(16), 5542-5549. https://doi.org/10.1128/AEM.00714-12
Mustaq, S., Moin, A., Pandit, B., Tiwary, B. K., & Alam, M. (2024). Phyllobacteriaceae: A family of ecologically and metabolically diverse bacteria with the potential for different applications. Folia Microbiologica, 69(1), 17-32. https://doi.org/10.1007/s12223-023-01107-2
Pagnani, G., Pellegrini, M., Galieni, A., D’Egidio, S., Matteucci, F., Ricci, A., Stagnari, F., Sergi, M., Lo Sterzo, C., Pisante, M., & Del Gallo, M. (2018). Plant growth-promoting rhizobacteria (PGPR) in Cannabis sativa ‘Finola’ cultivation: An alternative fertilization strategy to improve plant growth and quality characteristics. Industrial Crops and Products, 123, 75-83. https://doi.org/10.1016/j.indcrop.2018.06.033
Persmark, M., Pittman, P., Buyer, J. S., Schwyn, B., Gill, P. R., Jr., & Neilands, J. B. (1993). Isolation and structure of rhizobactin 1021, a siderophore from the alfalfa symbiont Rhizobium meliloti 1021. Journal of the American Chemical Society, 115(10), 3950-3956. https://doi.org/10.1021/ja00063a014
Poly, F., Ranjard, L., Nazaret, S., Gourbière, F., & Monrozier, L. J. (2001). Comparison of nifH gene pools in soils and soil microenvironments with contrasting properties. Applied and Environmental Microbiology, 67(5), 2255-2262. https://doi.org/10.1128/AEM.67.5.2255-2262.2001
Ponce de León, I., Oliver, J. P., Castro, A., Gaggero, C., Bentancor, M., & Vidal, S. (2007). Erwinia carotovora elicitors and Botrytis cinerea activate defense responses in Physcomitrella patens. BMC Plant Biology, 7, Article 52. https://doi.org/10.1186/1471-2229-7-52
Preacher, K. J. (2001, April). Calculation for the chi-square test: An interactive calculation tool for chi-square tests of goodness of fit and independence [Software]. https://quantpsy.org/chisq/chisq.htm
Reis, V. M., Olivares, F. L., & Döbereiner, J. (1994). Improved methodology for isolation of Acetobacter diazotrophicus and confirmation of its endophytic habitat. World Journal of Microbiology & Biotechnology, 10(4), 401-405. https://doi.org/10.1007/BF00144460
Rosenberg, E., & Zilber-Rosenberg, I. (2016). Microbes drive evolution of animals and plants: The hologenome concept. mBio, 7(2), Article e01395. https://doi.org/10.1128/mBio.01395-15
Sack, U., Hofrichter, M., & Fritsche, W. (1997). Degradation of polycyclic aromatic hydrocarbons by manganese peroxidase of Nematoloma frowardii. FEMS Microbiology Letters, 152(2), 227-234. https://doi.org/10.1111/j.1574-6968.1997.tb10432.x
Saikia, J., Mazumdar, R., & Thakur, D. (2022). Phylogenetic affiliation of endophytic actinobacteria associated with selected orchid species and their role in growth promotion and suppression of phytopathogens. Frontiers in Plant Science, 13, Article 1058867. https://doi.org/10.3389/fpls.2022.1058867
Sarwar, M., & Kremer, R. J. (1995). Determination of bacterially derived auxins using a microplate method. Letters in Applied Microbiology, 20(5), 282-285. https://doi.org/10.1111/j.1472-765X.1995.tb00446.x
Scheres, B., & van der Putten, W. H. (2017). The plant perceptron connects environment to development. Nature, 543(7645), 337-345. https://doi.org/10.1038/nature22010
Schwyn, B., & Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Analytical Biochemistry, 160(1), 47-56. https://doi.org/10.1016/0003-2697(87)90612-9
Scott, M., Rani, M., Samsatly, J., Charron, J. B., & Jabaji, S. (2018). Endophytes of industrial hemp (Cannabis sativa L.) cultivars: Identification of culturable bacteria and fungi in leaves, petioles, and seeds. Canadian Journal of Microbiology, 64(10), 664-680. https://doi.org/10.1139/cjm-2018-0108
Sylvester-Bradley, R., Asakawa, N., La Torraca, S. M., Magalhães, F. M. M., Oliveira, L. A., & Pereira, R. M. (1982). Levantamento quantitativo de microrganismos solubilizadores de fosfatos na rizosfera de gramíneas e leguminosas forrageiras na Amazônia. Acta Amazonica, 12(1), 15-22. https://doi.org/10.1590/1809-43921982121015
Taghinasab, M., & Jabaji, S. (2020). Cannabis microbiome and the role of endophytes in modulating the production of secondary metabolites: An overview. Microorganisms, 8(3), Article 355. https://doi.org/10.3390/microorganisms8030355
Taulé, C., Mareque, C., Barlocco, C., Hackembruch, F., Reis, V. M., Sicardi, M., & Battistoni, F. (2012). The contribution of nitrogen fixation to sugarcane (Saccharum officinarum L.), and the identification and characterization of part of the associated diazotrophic bacterial community. Plant and Soil, 356, 35-49. https://doi.org/10.1007/s11104-011-1023-4
Taulé, C., Zabaleta, M., Mareque, C., Platero, R., Sanjurjo, L., Sicardi, M., Frioni, L., Battistoni, F., & Fabiano, E. (2012). New betaproteobacterial Rhizobium strains able to efficiently nodulate Parapiptadenia rigida (Benth.) Brenan. Applied and Environmental Microbiology, 78(6), 1692-1700. https://doi.org/10.1128/AEM.06215-11
Uruguay XXI. (2024). Sector cannabis en Uruguay. https://www.uruguayxxi.gub.uy/uploads/informacion/0750cc9cb92a1af17373efce4443ee0d0be3538d.pdf
Vandenkoornhuyse, P., Quaiser, A., Duhamel, M., Le Van, A., & Dufresne, A. (2015). The importance of the microbiome of the plant holobiont. The New Phytologist, 206(4), 1196-1206. https://doi.org/10.1111/nph.13312
Verma, S. K., Kharwar, R. N., & White, J. F. (2019). The role of seed-vectored endophytes in seedling development and establishment. Symbiosis, 78(2), 107-113. https://doi.org/10.1007/s13199-019-00619-1
Wei, G., Ning, K., Zhang, G., Yu, H., Yang, S., Dai, F., Dong, L., & Chen, S. (2021). Compartment niche shapes the assembly and network of Cannabis sativa-associated microbiome. Frontiers in Microbiology, 12, Article 714993. https://doi.org/10.3389/fmicb.2021.714993
Winston, M. E., Hampton-Marcell, J., Zarraonaindia, I., Owens, S. M., Moreau, C. S., Gilbert, J. A., Hartsel, J. A., Kennedy, S. J., & Gibbons, S. M. (2014). Understanding cultivar-specificity and soil determinants of the cannabis microbiome. PloS ONE, 9(6), Article e99641. https://doi.org/10.1371/journal.pone.0099641
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