This study characterized 18 endophytic bacterial isolates in association with the root nodules of Leucaena leucocephala through phenotypic and genotypic analyses. The endophytes were associated with the plants and exhibited diverse plant growth-promoting (PGP) traits. Phosphate solubilization was observed in 39% of isolates at high levels and 33.3% at moderate levels. Siderophore production was prevalent, with 38.9% displaying high and 33.3% moderate production, aiding iron uptake. Indole-3-acetic acid (IAA) production varied (32.15 to 86.28 µg/ml) among the isolates. Notably, 94.4% of isolates showed positive hydrogen cyanide (HCN) production. Genetic diversity was assessed using the ARDRA clustered the isolates into eight morphotypes, whereas the phylogenetic analysis of the 16S rDNA sequences showed the presence of different genera including Rhizobium, Paenibacillus, Bacillus, Agrobacterium, Brucella, and Arthrobacter. On the other hand, these symbiotic endophytes are widely recognized for their mechanisms of plant growth promotion. Therefore, net house studies with rhizobial inoculation on L. leucocephala showed significant improvements in growth parameters such as shoot and root lengths, biomass, and nodulation, particularly with the strain Rhizobium sp. SoL9 (T3). Inoculation also enhanced soil properties, increasing nutrient availability and microbial populations. These endophytic bacterial isolates from L. leucocephala root nodules display genetic diversity and beneficial PGP traits, highlighting the potential for rhizobial biofertilization in enhancing plant development and soil fertility in legumes.

Center for Green Energy Research Career Point University Hamirpur 176041 India

Department of Basic Sciences Dr Yashwant Singh Parmar University of Horticulture and Forestry Nauni Solan Himachal Pradesh 173230 India

Department of Environmental Sciences Dr Yashwant Singh Parmar University of Horticulture and Forestry Nauni Solan Himachal Pradesh 173230 India

Department of Plant Pathology University of Horticulture and Forestry Dr Yashwant Singh Parmar Nauni Solan Himachal Pradesh 173230 India

Department of Soil Science and Water Management University of Horticulture and Forestry Dr Yashwant Singh Parmar Nauni Solan Himachal Pradesh 173230 India

Zobrazit více v PubMed

Adedayo AA, Babalola OO (2023) Genomic mechanisms of plant growth-promoting bacteria in the production of leguminous crops. Front Genet 14:1276003. https://doi.org/10.3389/fgene.2023.1276003 PubMed DOI PMC

Afzal I, Shinwari ZK, Sikandar S, Shahzad S (2019) Plant beneficial endophytic bacteria: mechanisms, diversity, host range and genetic determinants. Microbiol Res 221:36–49. https://doi.org/10.1016/j.micres.2019.02.001 PubMed DOI

Ahmad I, Suman K, Sharma B, Tewari L, Abulreesh, HH (2024) Deriving microbial community fingerprints from environmental samples using advanced molecular fingerprinting techniques. In S. Das & H. R. Dash (Eds.), Microbial diversity in the genomic era (2nd ed., pp. 133–145). Academic Press. https://doi.org/10.1016/B978-0-443-13320-6.00025-1

Ansari FA, Ahmad I, Khan AS (2024) Recent molecular tools for analyzing microbial diversity in rhizosphere ecosystem. In S. Das & H. R. Dash (Eds.), Microbial diversity in the genomic era (2nd ed., pp. 233–246). Academic Press. https://doi.org/10.1016/B978-0-443-13320-6.00007-X

Bakker AW, Schipper B (1987) Microbial cyanide production in the rhizosphere to potato yield reduction and Pseudomonas sp. mediated plant growth stimulation. Soil Biol Biochem 19(4):451–457. https://doi.org/10.1016/0038-0717(87)90037-X

Basu A, Prasad P, Das SN, Kalam S, Sayyed RZ, Reddy MS, El Enshasy H (2021) Plant growth promoting rhizobacteria (PGPR) as green bioinoculants: recent developments, constraints, and prospects. Sustainability 13(3):1140. https://doi.org/10.3390/su13031140 DOI

Battaglia ME, Martinez SI, Covacevich F, Consolo VF (2024) Trichoderma harzianum enhances root biomass production and promotes lateral root growth of soybean and common bean under drought stress. Ann Appl Biol 185:36–48. https://doi.org/10.1111/aab.12909 DOI

Chaudhary P, Agri U, Chaudhary A, Kumar A, Kumar G (2022) Endophytes and their potential in biotic stress management and crop production. Front Microbiol 13:933017. https://doi.org/10.3389/fmicb.2022.933017 PubMed DOI PMC

De Angelis A, Gasco L, Parisi G, Danieli PP (2021) A multipurpose leguminous plant for the Mediterranean countries: Leucaena leucocephala as an alternative protein source: a review. Animals 11(8):2230. https://doi.org/10.3390/ani11082230 PubMed DOI PMC

Debnath S, Chakraborty S, Langthasa M, Choure K, Agnihotri V, Srivastava A, Rai PK, Tilwari A, Maheshwari DK, Pandey P (2023) Non-rhizobial nodule endophytes improve nodulation, change root exudation pattern and promote the growth of lentil, for prospective application in fallow soil. Front Plant Sci. https://doi.org/10.3389/fpls.2023.1152875 PubMed DOI PMC

Dhiman VK, Rana N, Dhiman VK, Pandey H, Verma P, Singh D (2022) Effect of rhizobial isolates and nitrogen fertilizers on nursery performance, nodulation behavior, and nitrogenase activity of Dalbergia sissoo Roxb. seedlings. Plant Stress 4:100080. https://doi.org/10.1016/j.stress.2022.100080 DOI

Dimkić I, Janakiev T, Petrović M, Degrassi G, Fira D (2022) Plant-associated Bacillus and Pseudomonas antimicrobial activities in plant disease suppression via biological control mechanisms: a review. Physiol Mol Plant Pathol 117:101754. https://doi.org/10.1016/j.pmpp.2021.101754 DOI

Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797. https://doi.org/10.1093/nar/gkh340 PubMed DOI PMC

Edwards U, Rogall T, Blocker H, Emde M, Bottger EC (1989) Isolation and direct complete nucleotide determination of entire genes: characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17(19):7843–7853. https://doi.org/10.1093/nar/17.19.7843 PubMed DOI PMC

Etesami H, Glick BR (2024) Bacterial indole-3-acetic acid: a key regulator for plant growth, plant-microbe interactions, and agricultural adaptive resilience. Microbiol Res 281:127602. https://doi.org/10.1016/j.micres.2024.127602 PubMed DOI

Franco-Duarte R, Černáková L, Kadam S, Kaushik KS, Salehi B, Bevilacqua A, Corbo MR, Antolak H, Dybka-Stępień K, Leszczewicz M, Relison TS, Alexandrino de Souza VC, Sharifi-Rad J, Coutinho HDM, Martins N, Rodrigues CF (2019) Advances in chemical and biological methods to identify microorganisms-from past to present. Microorganisms 7(5):130. https://doi.org/10.3390/microorganisms7050130 PubMed DOI PMC

Gordon SA, Paleg LG (1957) Observation on the quantitative determination of indole acetic acid. Physiol Plant 10:39–47. https://doi.org/10.1111/j.1399-3054.1957.tb07608.x DOI

Goyal RK, Habtewold JZ (2023) Evaluation of legume–rhizobial symbiotic interactions beyond nitrogen fixation that help the host survival and diversification in hostile environments. Microorganisms 11(6):1454. https://doi.org/10.3390/microorganisms11061454 PubMed DOI PMC

Hall TA (1999) Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp 41:95–98

Hossain MS, Frith C, Bhattacharyya SS, DeLaune PB, Gentry TJ (2023) Isolation and characterization of bacterial endophytes from small nodules of field-grown peanut. Microorganisms 11(8):1941. https://doi.org/10.3390/microorganisms11081941 PubMed DOI PMC

Karasev ES, Hosid SL, Aksenova TS, Onishchuk OP, Kurchak ON, Dzyubenko NI, Andronov EE, Provorov NA (2023) Impacts of natural selection on evolution of core and symbiotically specialized (sym) genes in the polytypic species Neorhizobium galegae. Int J Mol Sci. https://doi.org/10.3390/ijms242316696 PubMed DOI PMC

Karthik C, Oves M, Sathya K, Sri RV, Arulselvi PI (2017) Isolation and characterization of multi-potential Rhizobium strain ND2 and its plant growth-promoting activities under Cr (VI) stress. Arch Agron Soil Sci 63:1058–1069. https://doi.org/10.1080/03650340.2016.1261116 DOI

Ketema P, Tefera T (2022) Effectiveness of rhizobium strains on faba bean (Vicia fabae L.) at Gumer district, highland area of Southern Ethiopia. Ukr J Ecol 12:13–18

Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16(2):111–120. https://doi.org/10.1007/BF01731581 PubMed DOI

Kiruba NJM, Thatheyus AJ (2021) Fungi, fungal enzymes and their potential application as biostimulants. In J. White, A. Kumar, & S. Droby (Eds.), Microbiome Stimulants for Crops (pp. 305–314). Woodhead Publishing. https://doi.org/10.1016/B978-0-12-822122-8.00024-8

Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35(6):1547–1549 PubMed DOI PMC

Kumar S, Singh M, Kumar S, Rajeev (2024) Changes in root nodules dynamics under mixed cropping with varying nutrient management of berseem crop and succeeding fodder cowpea. J Plant Nutr 48:617–638. https://doi.org/10.1080/01904167.2024.2410818

Ladha JK, Peoples MB, Reddy PM, Biswas JC, Bennett A, Jat ML, Krupnik TJ (2022) Biological nitrogen fixation and prospects for ecological intensification in cereal-based cropping systems. Field Crops Res 283:108541. https://doi.org/10.1016/j.fcr.2022.108541 PubMed DOI PMC

Luo Y, Ma L, Feng Q, Luo H, Chen C, Wang S, Yuan Y, Liu C, Cao X, Li N (2024) Influence and role of fungi, bacteria, and mixed microbial populations on phosphorus acquisition in plants. Agriculture (Basel) 14(3):358. https://doi.org/10.3390/agriculture14030358 DOI

Ma W, Tang S, Dengzeng Z, Zhang D, Zhang T, Ma X (2022) Root exudates contribute to belowground ecosystem hotspots: a review. Front Microbiol 13:937940. https://doi.org/10.3389/fmicb.2022.937940 PubMed DOI PMC

Menge DNL, Wolf AA, Funk JL, Perakis SS, Akana PR, Arkebauer R, Bytnerowicz T (2023) Tree symbioses sustain nitrogen fixation despite excess nitrogen supply. Ecol Monogr 93(2):e1562. https://doi.org/10.1002/ecm.1562 DOI

Mervin HD, Peech M (1951) Exchangeability of soil potassium in the sand, silt and clay fraction as influenced by the nature of the complementary exchangeable cation. Soil Sci Soc Am Proc 15:125–128. https://doi.org/10.2136/sssaj1951.036159950015000C0026x DOI

Miljaković D, Marinković J (2024) Harnessing plant growth-promoting rhizobacteria: a dual approach as biofertilizers and biopesticides for field and vegetable crop production. In: Dheeman S, Islam MT, Egamberdieva D, Siddiqui MN (eds) Soil Bacteria. Springer, Singapore. https://doi.org/10.1007/978-981-97-3473-3_15

Olsen SR, Cole CV, Watanabe FS, Dean LA (1954) Estimation of available phosphorus in soils by extraction with sodium bicarbonate. USDA Circular No. 939. US Government Printing Office, Washington, DC

Pattnaik S, Mohapatra B, Gupta A (2021) Plant growth-promoting microbe mediated uptake of essential nutrients (Fe, P, K) for crop stress management: microbe–soil–plant continuum. Front Agron 3:689972. https://doi.org/10.3389/fagro.2021.689972 DOI

Pervin S, Jannat B, Sanjee S, Farzana T (2017) Characterization of rhizobia from root nodule and rhizosphere of Lablab purpureus and Vigna sinensis in Bangladesh. Turkish JAF Sci Tech 5(1):14–17. https://doi.org/10.24925/turjaf.v5i1.14-17.743

Pikovskaya RI (1948) Solubilization of phosphorus in soil in connection with vital activity of some microbial species. Microbiology 17:362–370

Rafique M, Naveed M, Mumtaz MZ (2024) Unlocking the potential of biofilm-forming plant growth-promoting rhizobacteria for growth and yield enhancement in wheat (Triticum aestivum L.). Sci Rep 14:15546. https://doi.org/10.1038/s41598-024-66562-4 PubMed DOI PMC

Rao NSS (1977) Soil microorganisms and plant growth. 3rd ed., rev. and enl. Oxford & IBH Publishing Co, New Delhi.

Rathod K, Patel A, Mulani R (2023) Isolation, characterization and molecular identification of Rhizobium spp. and Trichoderma spp. from nodule and rhizospheric soil of Arachis hypogaea. Acta sci microbiol 6(12). https://doi.org/10.31080/ASMI.2023.06.1319

Saharan BS, Nehra V (2011) Plant growth promoting rhizobacteria: a critical review. J Life Sci Med Res 21:1–300

Sambrook J, Fritsch ER, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophore. Anal Biochem 160(1):751–759. https://doi.org/10.1016/0003-2697(87)90612-9

Sharma P, Kaur A, Batish DR, Kaur S, Chauhan BS (2022) Critical insights into the ecological and invasive attributes of Leucaena leucocephala, a tropical agroforestry species. Front Agron 4:890992. https://doi.org/10.3389/fagro.2022.890992 DOI

Singh S, Dutta U, Bhat AK, Gupta S, Gupta V, Jamwal S (2017) Morpho-cultural and biochemical identification of Pseudomonas sp. isolated from the rhizosphere of different vegetable crops and study its efficacy on Solanum melongena (brinjal). J Pharmacogn Phytochem 6(2):22–28

Subbiah BV, Asija CL (1956) A rapid procedure for estimation of available nitrogen in soils. Curr Sci 25:259–260

Sunitha Kumari K, Padma Devi SN, Ranjithkumar R, Djearamane S, Tey LH, Wong LS, Kayarohanam S, Arumugam N, Almansour AI, Perumal K (2023) Organic remobilization of zinc and phosphorus availability to plants by application of mineral solubilizing bacteria Pseudomonas aeruginosa. Heliyon 9(11):e22128. https://doi.org/10.1016/j.heliyon.2023.e22128 PubMed DOI PMC

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725–2729. https://doi.org/10.1093/molbev/mst197 PubMed DOI PMC

Thakur N, Kumari P, Sharma H, Kumari G (2023) Tracking of soil microbial inoculates for sustainable agriculture. In S. Gangola, S. Kumar, S. Joshi, & P. Bhatt (Eds.), Developments in applied microbiology and biotechnology: Advanced microbial technology for sustainable agriculture and environment (pp. 155–173). Academic Press. https://doi.org/10.1016/B978-0-323-95090-9.00012-1

Vincent JM (1970) A manual for the practical study of root nodule bacteria. Blackwell Scientific Publishers, Oxford, p 164. https://doi.org/10.1002/jobm.19720120524

Walkley A, Black IA (1934) An examination of DEGTJAREFF method of determining soil organic matter and a proposed modification of chromic and titration method. Soil Sci 37:29–38. https://doi.org/10.1097/00010694-193401000-00003 DOI

Wilson DO, Reisenauer HM (1963) Determination of leghemoglobin in legume nodules. Anal Biochem 6(1):27–30. https://doi.org/10.1016/0003-2697(63)90004-6 DOI

Zhou Y, Zhu H, Fu S, Yao Q (2017) Variation in soil microbial community structure associated with different legume species is greater than that associated with different grass species. Front Microbiol 8:1–13. https://doi.org/10.3389/fmicb.2017 DOI