Novel Cell-Free Suspensions of Symbiotic Bacteria for Biocontrol of Phytopathogenic Bacteria

(Marwa Asad Nayef and Najwa Ibrahim Khaleel Al-Barhawee)

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Abstract

Biofertilizers utilize plant growth-promoting bacteria (PGPB), which are symbiotic bacteria found in the root nodules of leguminous plants that fix atmospheric nitrogen. This study utilized cell-free bacterial suspensions from several leguminous plants, including Vicia faba, Trifolium repens, Lens culinaris, Trigonella foenum-graecum, Lens culinaris subsp. orientalis and Medicago sativa. Six isolates (MA1–MA6) were tested as biocontrol agents against Agrobacterium tumefaciens RB04, Lelliottia amnigena MN1 and Xanthomonas campestris. After establishing that these bacteria are pathogenic, we evaluated their antagonistic activity using the cross method. This identified MA2 as the most effective isolate, with MA5 following closely. Among the tested isolates, MA2 was the most successful in inhibiting the growth of pathogenic bacteria, followed by MA5. The other isolates only impacted the growth of two bacterial species: L. amnigena strain MN1 and X campestris. The agar well diffusion method showed bacterial sensitivity to cell-free supernatants (CFSs) at 67%, 34% and 17% for A. tumefaciens, X. campestris and L. amnigena, respectively. CFS MA6, the most effective supernatant, was analyzed via gas chromatography-mass spectrometry, revealing four active compounds: C7H13NO2, C11H18N2O2, C28H53NO3 and C21H39NO3, with molecular weights of 143, 210, 451 and 353, respectively.  
KEYWORDS
Agrobacterium tumefaciens, antagonistic, biofertilizers, GC-MS, legume plants, PGPB

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References

Al-Ani, R.A., Adhab, M.A., Mahdi, M.H. and Abood, H.M. (2012). Rhizobium japonicum as a biocontrol agent of soybean root rot disease caused by fusarium solani and macrophomina phaseolina. Plant Protection Science, 48(4), 149. DOI: 10.17221/16/2012-PPS.
Al-Barhawee, N.I.K. and Al-Rubyee, S.S. (2024). Inhibition of biofilm formation in Agrobacterium tumefaciens by cell-free supernatants of Pseudomonas aeruginosa analyzed by GC-MS. Baghdad Science Journal, 21(7), 2222–36. DOI: 10.21123/bsj.2023.8692.
Danneels, B., Pinto-Carbó, M. and Carlier, A. (2018). Patterns of nucleotide deletion and insertion inferred from bacterial pseudogenes. Genome Biology and Evolution, 10(7), 1792–802. DOI: 10.1093/gbe/evy140.
Egamberdieva, D., Wirth, S.J., Alqarawi, A.A., Abd_Allah, E.F. and Hashem, A. (2017). Phytohormones and beneficial microbes: Essential components for plants to balance stress and fitness. Frontiers in Microbiology, 8(n/a), 2104. DOI: 10.3389/fmicb.2017.02104.
El-Mokhtar, M.A., Hassanein, K.M., Ahmed, A.S., Gad, G.F., Amin, M.M. and Hassanein, O.F. (2020). Antagonistic activities of cell-free supernatants of lactobacilli against extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Pseudomonas aeruginosa. Infection and Drug Resistance, n/a(n/a)543–52. DOI: 10.2147/IDR.S235603.
Esteban-Herrero, G., Álvarez, B., Santander, R.D. and Biosca, E.G. (2023). Screening for novel beneficial environmental bacteria for an antagonism-based biological control. Microorganisms, 11(7), 1795. DOI: 10.3390/microorganisms11071795.
Gaurav, A., Bakht, P., Saini, M., Pandey, S. and Pathania, R. (2023). Role of bacterial efflux pumps in antibiotic resistance, virulence and strategies to discover novel efflux pump inhibitors. Microbiology, 169(5), 001333. DOI: 10.1099/mic.0.001333.
Gleckman, R., Blagg, N. and Joubert, D.W. (1981). Trimethoprim: mechanisms of action, antimicrobial activity, bacterial resistance, pharmacokinetics, adverse reactions and therapeutic indications. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy, 1(1), 14–9. DOI: 10.1002/j.1875-9114.1981.tb03548.x.
Goad, C.L. (2020). SAS Programming for Elementary Statistics: Getting Started. 1st Edition. Chapman and Hall/CRC. DOI: 10.1201/9780429491900
Gopal, M.A. and Thirupathi, A. (2020). Isolation and identification of Pseudomonas aeruginosa FROM UROPATHOGENS. University of Yangon Research Journal, 7(11), n/a.
Hefetz, I., Israeli, O., Bilinsky, G., Plaschkes, I., Hazkani-Covo, E., Hayouka, Z. and Helman, Y. (2023). A reversible mutation in a genomic hotspot saves bacterial swarms from extinction. Iscience, 26(2). 106043 DOI: 10.1016/j.isci.2023.106043.
Hooykaas, P.J. (2023). The Ti plasmid, driver of Agrobacterium pathogenesis. Phytopathology, 113(4), 594–604. DOI: 10.1094/PHYTO-11-22-0432-IA.
Horton, J.S. and Taylor, T.B. (2023). Mutation bias and adaptation in bacteria. Microbiology, 169(11), 001404. DOI: 10.1099/mic.0.001404.
Iglesias-Bernabé, L., Madloo, P., Rodríguez, V.M., Francisco, M. and Soengas, P. (2019). Dissecting quantitative resistance to Xanthomonas campestris pv. campestris in leaves of Brassica oleracea by QTL analysis. Scientific Reports, 9(1), n/a. DOI: 10.1038/s41598-019-38527-5.
Islam, M.S., Akter, M.M., Rahman, M.A., Rahman, M.M., Akhtar, M. and Alam, M.F. (2010). Isolation of Agrobacterium tumefaciens strains from crown gall sample of dicot plants in Bangladesh. Current Research in Bacteriology, 3(n/a), 27–36. DOI: 10.3923/crb.2010.27.36.
Jumaah, O., Sultan, R. and Assafi, M. (2022). Antimicrobial activity of local rhizobial isolates against some fungi. Journal of Education and Science, 31(2), 141–9. DOI: 10.33899/edusj.2022.133511.1230.
Kiran, G.S., Priyadharsini, S., Sajayan, A., Ravindran, A. and Selvin, J. (2018). An antibiotic agent pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro isolated from a marine bacteria Bacillus tequilensis MSI45 effectively controls multi-drug-resistant Staphylococcus aureus. RSC Advances, 8(32), 17837–46. DOI: 10.1039/C8RA00820E.
Li, J.H., Qin, S.Y., Shi, Y.X., Xie, X.W., Chai, A.L., Wang, Y.H. and Li, L. (2025). First report of Lelliottia amnigena causing soft rot on purple stem mustards in China. Plant Disease, 109(1), 228. DOI: 10.1094/PDIS-01-24-0180-PDN.
Liang, J.H. and Han, X. (2013). Structure-activity relationships and mechanism of action of macrolides derived from erythromycin as antibacterial agents. Current Topics in Medicinal Chemistry, 13(24), 3131–64. DOI: 10.2174/15680266113136660223.
Long, H., Sung, W., Miller, S.F., Ackerman, M.S., Doak, T.G. and Lynch, M. (2015). Mutation rate, spectrum, topology and context-dependency in the DNA mismatch repair-deficient Pseudomonas fluorescens ATCC948. Genome Biology and Evolution, 7(1), 262–71. DOI: 10.1093/gbe/evu284.
Lynch, M. (2006). Streamlining and simplification of microbial genome architecture. Annual Review of Microbiology, 60(1), 327–49. DOI: 10.1146/annurev.micro.60.080805.142300.
Mokrani, S., Nabti, E.H. and Cruz, C. (2020). Current advances in plant growth promoting bacteria alleviating salt stress for sustainable agriculture. Applied Sciences, 10(20), 7025. DOI:10.3390/app10207025.
Morcillo, R.J.L., Baroja-Fernández, E., López-Serrano, L., Leal-López, J., Muñoz, F.J., Bahaji, A. and Pozueta-Romero, J. (2022). Cell-free microbial culture filtrates as candidate biostimulants to enhance plant growth and yield and activate soil-and plant-associated beneficial microbiota. Frontiers in Plant Science, 13(n/a), 1040515.   DOI: 10.3389/fpls.2022.1040515.
Naseem, H., Ahsan, M., Shahid, M.A. and Khan, N. (2018). Exopolysaccharides produce rhizobacteria and their role in plant growth and drought tolerance. Journal of Basic Microbiology, 58(12), 1009–22. DOI: 10.1002/jobm.201800309.
Nilsson, A.I., Koskiniemi, S., Eriksson, S., Kugelberg, E., Hinton, J.C.D. and Andersson, D.I. (2005). Bacterial genome size reduction by experimental evolution. Proceedings of the National Academy of Sciences, 102(34), 12112–6. DOI: 10.1073/pnas.0503654102.
Osei, R., Yang, C., Cui, L., Ma, T., Li, Z. and Boamah, S. (2022). Isolation, identification and pathogenicity of Lelliottia amnigena causing soft rot of potato tuber in China. Microbial Pathogenesis, 164(6), 105441. DOI: 10.1016/j.micpath.2022.105441.
Pantigoso, H.A., Newberger, D. and Vivanco, J.M. (2022). The rhizosphere microbiome: Plant–microbial interactions for resource acquisition. Journal of Applied Microbiology, 133(5), 2864–76. DOI: 10.1111/jam.15686.
Payne, J.L., Menardo, F., Trauner, A., Borrell, S., Gygli, S.M., Loiseau, C. and Hall, A.R. (2019). Transition bias influences the evolution of antibiotic resistance in Mycobacterium tuberculosis. PLoS Biology, 17(5), e3000265. DOI: 10.1371/journal.pbio.3000265
Pellegrini, M., Pagnani, G., Bernardi, M., Mattedi, A., Spera, D.M. and Gallo, M.D. (2020). Cell-free supernatants of plant growth-promoting bacteria: a review of their use as biostimulant and microbial biocontrol agents in sustainable agriculture. Sustainability, 12(23), 9917.  DOI: 10.3390/su12239917.
Popović, T., Jošić, D., Starović, M., Milovanović, P., Dolovac, N., Poštić, D. and Stanković, S. (2013). Phenotypic and genotypic characterization of Xanthomonas campestris strains isolated from cabbage, kale and broccoli. Archives of Biological Sciences, 65(2), 585–93. DOI: 10.2298/ABS1302585P.
Rashad, Y.M., Al Tami, M.S. and Abdalla, S.A. (2023). Eliciting transcriptomic and antioxidant defensive responses against Rhizoctonia root rot of sorghum using the endophyte Aspergillus oryzae YRA3. Scientific Reports, 13(1), 19823. DOI: 10.1038/s41598-023-46696-7.
Riseh, R.S., Vatankhah, M., Hassanisaadi, M. and Ait Barka, E. (2024). Unveiling the role of hydrolytic enzymes from soil biocontrol bacteria in sustainable phytopathogen management. Frontiers in Bioscience-Landmark, 29(3), 105.  DOI: 10.31083/j.fbl2903105.
Rudolf, I., Mendel, J., Šikutová, S., Švec, P., Masaříková, J., Nováková, D. and Hubálek, Z. (2009). 16S rRNA gene-based identification of cultured bacterial flora from host-seeking Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna ticks, vectors of vertebrate pathogens. Folia Microbiologica, 54(5), 419–28.  DOI: 10.1007/s12223-009-0059-9.
Selveshwari, S., Lele, K. and Dey, S. (2021). Genomic signatures of UV resistance evolution in Escherichia coli depend on the growth phase during exposure. Journal of Evolutionary Biology, 34(6), 953–67.  DOI: 10.1111/jeb.13764.
Ser, H.L., Palanisamy, U.D., Yin, W.F., Abd Malek, S.N., Chan, K.G., Goh, B.H. and Lee, L.H. (2015). Presence of antioxidative agent, pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-in newly isolated Streptomyces mangrovisoli sp. nov. Frontiers in Microbiology, 6(n/a), 854. DOI: 10.3389/fmicb.2015.00854.
Skowronek, M., Sajnaga, E., Pleszczyńska, M., Kazimierczak, W., Lis, M. and Wiater, A. (2020). Bacteria from the midgut of common cockchafer (Melolontha melolontha L.) larvae exhibiting antagonistic activity against bacterial symbionts of entomopathogenic nematodes: isolation and molecular identification. International Journal of Molecular Sciences, 21(2), 580.  DOI: 10.3390/ijms21020580.
Soriful, I.M., Akter, M.M., Rahman, M.M., Akhtar, M.M. and Alam, M.F. (2010). Isolation of Agrobacterium tumefaciens strains from crown gall sample of dicot plants in Bangladesh. Current Research in Bacteriology, 3(1), 27–36.  DOI: 10.3923/crb.2010.27.36.
Spagnolo, F., Rinaldi, C., Sajorda, D.R. and Dykhuizen, D.E. (2016). Evolution of resistance to continuously increasing streptomycin concentrations in populations of Escherichia coli. Antimicrobial Agents and Chemotherapy, 60(3), 1336–42. DOI: 10.1128/aac.01359-15.
Tariq, M., Khan, A., Asif, M., Khan, F., Ansari, T., Shariq, M. and Siddiqui, M.A. (2020). Biological control: a sustainable and practical approach for plant disease management. Acta Agriculture Scandinavica, Section B-Soil and Plant Science, 70(6), 507–24. DOI: 10.1080/09064710.2020.1784262.
Trigui, F., Pigeon, P., Jalleli, K., Top, S., Aifa, S. and El Arbi, M. (2013). Selection of a suitable disc bioassay for the screening of anti-tumor molecules. International Journal of Biomedical Science, 9(4), 230. DOI: 10.59566/IJBS.2013.9230.
Vivekanandan, K.E., Kumar, P.V., Jaysree, R.C. and Rajeshwari, T. (2025). Exploring molecular mechanisms of drug resistance in bacteria and progressions in crispr/cas9-based genome expurgation solutions. Global Medical Genetics, 12(2) 100042. DOI:10.1016/j.gmg.2025.100042.
Wu, H., Guo, T., Yang, S., Guo, Z., Kang, B., Liu, L. and Peng, B. (2023). First report of bacterial soft rot caused by Enterobacter mori affecting host watermelon. Plant Disease, 107(7), 2209. DOI:10.1094/PDIS-05-22-1048-PDN.
Yu, T. and Zeng, F. (2024). Chloramphenicol interferes with 50s ribosomal subunit maturation via direct and indirect mechanisms. Biomolecules, 14(10), 1225. DOI: 10.3390/biom14101225.