Soil DNA studies often present a misleading view of microbial communities by failing to distinguish between live and dead bacterial cells. There is a critical gap in research on how the composition of dead cells diverges from that of living ones and which ecological factors drive these differences. Understanding the live/dead distinction is essential for gaining insights into soil biological activity and nutrient reservoirs. In this study, we employed flow cytometry with fluorescent labeling, which allowed us to obtain single-cell measurements from large samples and effectively differentiate between live and dead bacteria, focusing on the impact of proximity to desert plants on soil microbial communities. We specifically investigated three desert plants— Zygophyllum dumosum , Hammada scoparia , and Atriplex halimus —and compared their influence to that of soil crust and bare soil. Our findings reveal that Proteobacteria and Actinobacteria phyla are abundant in both live and dead fractions. The diversity of live bacterial communities is significantly higher in samples near H. scoparia and A. halimus compared to Z. dumosum and soil crust. Each plant species forms distinct nutrient islands, which markedly influence bacterial composition and the live-to-dead cell ratio. Importantly, living bacterial cells are less abundant in soil crust and bare ground compared to those beneath H. scoparia and A. halimus . The nutrient-poor conditions of bare soil favor communities of phyla known for their long-term ecological persistence. Conversely, Z. dumosum correlates with a higher proportion of dead cells, underscoring a paradox in its influence on microbial dynamics. In conclusion, the live/dead ratio alone does not provide a complete picture of ecological processes. Our results assert the necessity of looking beyond total bacterial communities to comprehensively understand the key contributors to soil ecology and function.
KeywordsRhizosphereMicrobial CommunityLive/Dead BacteriaPlantsArid System
Davey, H.M. (2011) Life, Death, and In-Between: Meanings and Methods in Microbiology. Applied and Environmental Microbiology , 77, 5571-5576.
Insam, H., Parkinson, D. and Domsch, K.H. (1989) Influence of Macroclimate on Soil Microbial Biomass. Soil Biology and Biochemistry , 21, 211-221. https://doi.org/10.1016/0038-0717(89)90097-7
Whitford, W.G. and Duval, B.D. (2019) Ecology of Desert Systems. Academic Press.
Smith, J. and Paul, E. (1990) The Significance of Microbial Biomass in the Carbon and Nitrogen Dynamics of Soil. Soil Biochemistry , 6, 415-471.
Steinhaus, E.A. and Birkeland, J.M. (1939) Studies on the Life and Death of Bacteria: I. The Senescent Phase in Aging Cultures and the Probable Mechanisms Involved. Journal of Bacteriology , 38, 249-261. https://doi.org/10.1128/jb.38.3.249-261.1939
Jansson, J.K. and Hofmockel, K.S. (2019) Soil Microbiomes and Climate Change. Nature Reviews Microbiology , 18, 35-46. https://doi.org/10.1038/s41579-019-0265-7
Davey, H.M. (2011) Life, Death, and In-Between: Meanings and Methods in Microbiology. Applied and Environmental Microbiology , 77, 5571-5576. https://doi.org/10.1128/aem.00744-11
Emerson, J.B., Adams, R.I., Román, C.M.B., Brooks, B., Coil, D.A., Dahlhausen, K., et al. (2017) Schrödinger’s Microbes: Tools for Distinguishing the Living from the Dead in Microbial Ecosystems. Microbiome , 5, Article No. 86. https://doi.org/10.1186/s40168-017-0285-3
Keller, M. and Zengler, K. (2004) Tapping into Microbial Diversity. Nature Reviews Microbiology , 2, 141-150. https://doi.org/10.1038/nrmicro819
Mukamolova, G.V., Murzin, A.G., Salina, E.G., Demina, G.R., Kell, D.B., Kaprelyants, A.S., et al. (2005) Muralytic Activity of Micrococcus luteus RPF and Its Relationship to Physiological Activity in Promoting Bacterial Growth and Resuscitation. Molecular Microbiology , 59, 84-98. https://doi.org/10.1111/j.1365-2958.2005.04930.x
PostGate, J.R. (1967) Viability Measurements and the Survival of Microbes under Minimum Stress. Advances in Microbial Physiology , 1, 1-23. https://doi.org/10.1016/s0065-2911(08)60248-9
Wayne, L.G. (1994) Dormancy of Mycobacterium tuberculosis and Latency of Disease. European Journal of Clinical Microbiology & Infectious Diseases , 13, 908-914. https://doi.org/10.1007/bf02111491
Jones, R.P. (1987) Factors Influencing Deactivation of Yeast Cells Exposed to Ethanol. Journal of Applied Bacteriology , 63, 153-164. https://doi.org/10.1111/j.1365-2672.1987.tb02698.x
Humaira, Z., Mirza, I.A., Fayyaz, M. and Hussain, W. (2022) Application of Flow-Cytometry in a Field of Microbiology: A New Horizon for Rapid Microbial Diagnosis. Journal of Clinical Obstetrics and Gynecology , 4, Article 1.
Ou, F., McGoverin, C., White, J., Swift, S. and Vanholsbeeck, F. (2019) Bead-Based Flow-Cytometric Cell Counting of Live and Dead Bacteria. In: Iovino, F., Ed., Streptococcus Pneumonia e , Springer, 123-134. https://doi.org/10.1007/978-1-4939-9199-0_11
Winson, M.K. and Davey, H.M. (2000) Flow Cytometric Analysis of Microorganisms. Methods , 21, 231-240. https://doi.org/10.1006/meth.2000.1003
Shamir, I., Zahavy, E. and Steinberger, Y. (2009) Bacterial Viability Assessment by Flow Cytometry Analysis in Soil. Frontiers of Biology in China , 4, 424-435. https://doi.org/10.1007/s11515-009-0050-5
Porter, J. (1997) Go with the Flow—Use of Flow Cytometry in Environmental Microbiology. FEMS Microbiology Ecology , 24, 93-101. https://doi.org/10.1016/s0168-6496(97)00038-x
Sarig, S. and Steinberger, Y. (1994) Microbial Biomass Response to Seasonal Fluctuation in Soil Salinity under the Canopy of Desert Halophytes. Soil Biology and Biochemistry , 26, 1405-1408. https://doi.org/10.1016/0038-0717(94)90224-0
Sarig, S., Fliessbach, A. and Steinberger, Y. (1999) Soil Microbial Biomass under the Canopy of Coastal Sand Dune Shrubs. Arid Soil Research and Rehabilitation , 13, 75-80. https://doi.org/10.1080/089030699263500
Shamir, I. and Steinberger, Y. (2007) Vertical Distribution and Activity of Soil Microbial Population in a Sandy Desert Ecosystem. Microbial Ecology , 53, 340-347. https://doi.org/10.1007/s00248-006-9137-6
Belnap, J. (2003) The World at Your Feet: Desert Biological Soil Crusts. Frontiers in Ecology and the Environment , 1, 181-189. https://doi.org/10.1890/1540-9295(2003)001[0181:twayfd]2.0.co;2
Saul‐Tcherkas, V. and Steinberger, Y. (2009) Temporal and Shrub Adaptation Effect on Soil Microbial Functional Diversity in a Desert System. European Journal of Soil Science , 60, 871-882. https://doi.org/10.1111/j.1365-2389.2009.01165.x
Berg, N. and Steinberger, Y. (2008) Role of Perennial Plants in Determining the Activity of the Microbial Community in the Negev Desert Ecosystem. Soil Biology and Biochemistry , 40, 2686-2695. https://doi.org/10.1016/j.soilbio.2008.07.019
Yu, J. and Steinberger, Y. (2011) Vertical Distribution of Microbial Community Functionality under the Canopies of Zygophyllum dumosum and Hammada Scoparia in the Negev Desert, Israel. Microbial Ecology , 62, 218-227. https://doi.org/10.1007/s00248-011-9846-3
Noy-Meir, I. (1973) Desert Ecosystems: Environment and Producers. Annual Review of Ecology and Systematics , 4, 25-51. https://doi.org/10.1146/annurev.es.04.110173.000325
Shmueli, I., Barness, G. and Steinberger, Y. (2007) Soil Microbial Population in the Vicinity of the Bean Caper ( Zygophyllum dumosum ) Root Zone in a Desert System. Pedosphere , 17, 758-765. https://doi.org/10.1016/s1002-0160(07)60091-5
Sherman, C., Marais, E., Maggs-Kölling, G., Adams, J. and Steinberger, Y. (2019) Abiotic and Plant Gender Effects on the Structure and Function of Soil Microbial Communities Associated with Acanthosicyos horridus (Nara) in the Namibian Sand-Dune Desert Ecosystem. Journal of Arid Environments , 163, 50-58. https://doi.org/10.1016/j.jaridenv.2019.01.009
Doniger, T., Adams, J.M., Marais, E., Maggs-Kölling, G., Sherman, C., Kerfahi, D., et al. (2020) The ‘Fertile Island Effect’ of Welwitschia Plants on Soil Microbiota Is Influenced by Plant Gender. FEMS Microbiology Ecology , 96, fiaa186. https://doi.org/10.1093/femsec/fiaa186
Sokol, N.W., Slessarev, E., Marschmann, G.L., Nicolas, A., Blazewicz, S.J., Brodie, E.L., et al. (2022) Life and Death in the Soil Microbiome: How Ecological Processes Influence Biogeochemistry. Nature Reviews Microbiology , 20, 415-430. https://doi.org/10.1038/s41579-022-00695-z
Evenari, M., Shanan, L. and Tadmor, N. (1982) The Negev: The Challenge of a Desert. Harvard University Press.
Barness, G., Rodriguez Zaragoza, S., Shmueli, I. and Steinberger, Y. (2008) Vertical Distribution of a Soil Microbial Community as Affected by Plant Ecophysiological Adaptation in a Desert System. Microbial Ecology , 57, 36-49. https://doi.org/10.1007/s00248-008-9396-5
Yu, J. and Steinberger, Y. (2012) Vertical Distribution of Soil Microbial Biomass and Its Association with Shrubs from the Negev Desert. Journal of Arid Environments , 78, 110-118. https://doi.org/10.1016/j.jaridenv.2011.11.012
Martirosyan, V. and Steinberger, Y. (2014) Microbial Functional Diversity in the Phyllosphere and Laimosphere of Different Desert Plants. Journal of Arid Environ ments , 107, 26-33. https://doi.org/10.1016/j.jaridenv.2014.04.002
Kaplan, D., Maymon, M., Agapakis, C.M., Lee, A., Wang, A., Prigge, B.A., et al. (2013) A Survey of the Microbial Community in the Rhizosphere of Two Dominant Shrubs of the Negev Desert Highlands, Zygophyllum dumosum (Zygophyllaceae) and Atriplex halimus (Amaranthaceae), Using Cultivation-Dependent and Cultivation-Independent Methods. American Journal of Botany , 100, 1713-1725. https://doi.org/10.3732/ajb.1200615
Waqar, S., Bhat, A.A. and Khan, A.A. (2024) Endophytic Fungi: Unravelling Plant-Endophyte Interaction and the Multifaceted Role of Fungal Endophytes in Stress Amelioration. Plant Physiology and Biochemistry , 206, Article ID: 108174. https://doi.org/10.1016/j.plaphy.2023.108174
Dekic, S., Hrenovic, J., Durn, G. and Venter, C. (2020) Survival of Extensively-and Pandrug-Resistant Isolates of Acinetobacter baumannii in Soils. Applied Soil Ecology , 147, Article ID: 103396. https://doi.org/10.1016/j.apsoil.2019.103396
Choi, J., Kim, M., Roh, S.W. and Bae, J. (2010) Acidovorax Soli sp. nov., Isolated from Landfill Soil. International Journal of Systematic and Evolutionary Microbiology , 60, 2715-2718. https://doi.org/10.1099/ijs.0.019661-0
Wilhelm, R.C., Singh, R., Eltis, L.D. and Mohn, W.W. (2018) Bacterial Contributions to Delignification and Lignocellulose Degradation in Forest Soils with Metagenomic and Quantitative Stable Isotope Probing. The ISME Journal , 13, 413-429. https://doi.org/10.1038/s41396-018-0279-6
Tzfira, T. and Citovsky, V. (2007) Agrobacterium : From Biology to Biotechnology. Springer Science & Business Media. https://doi.org/10.1007/978-0-387-72290-0
Walker, T.S., Bais, H.P., Déziel, E., Schweizer, H.P., Rahme, L.G., Fall, R., et al. (2004) Pseudomonas aeruginosa -Plant Root Interactions. Pathogenicity, Biofilm Formation, and Root Exudation. Plant Physiology , 134, 320-331. https://doi.org/10.1104/pp.103.027888
Yang, L., Danzberger, J., Schöler, A., Schröder, P., Schloter, M. and Radl, V. (2017) Dominant Groups of Potentially Active Bacteria Shared by Barley Seeds Become Less Abundant in Root Associated Microbiome. Frontiers in Plant Science , 8, Article 1005. https://doi.org/10.3389/fpls.2017.01005
Sah, S., Krishnani, S. and Singh, R. (2021) Pseudomonas Mediated Nutritional and Growth Promotional Activities for Sustainable Food Security. Current Research in Microbial Sciences , 2, Article ID: 100084. https://doi.org/10.1016/j.crmicr.2021.100084
Smith, J., Johnson, A. and Brown, K. (2020) Bacterial Diversity in Soil Samples beneath Different Vegetation Types. Journal of Soil Science , 35, 123-135.
Bogosian, G. and Bourneuf, E.V. (2001) A Matter of Bacterial Life and Death. EMBO reports , 2, 770-774. https://doi.org/10.1093/embo-reports/kve182
Steen, H.B. and Boye, E. (1980) Bacterial Growth Studied by Flow Cytometry. Cytometry , 1, 32-36. https://doi.org/10.1002/cyto.990010108
Mushtaq, H., Ganai, B.A. and Jehangir, A. (2023) Exploring Soil Bacterial Diversity in Different Micro-Vegetational Habitats of Dachigam National Park in North-Western Himalaya. Scientific Reports , 13, Article No. 3090. https://doi.org/10.1038/s41598-023-30187-w