Functional Characterization of a Novel Bacillus amyloliquefaciens and Halomonas elongata Consortium for Mitigating Fusarium Crown Rot and Salinity Stress in Wheat

Functional Characterization of a Novel Bacillus amyloliquefaciens and Halomonas elongata Consortium for Mitigating Fusarium Crown Rot and Salinity Stress in Wheat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Functional Characterization of a Novel Bacillus amyloliquefaciens and Halomonas elongata Consortium for Mitigating Fusarium Crown Rot and Salinity Stress in Wheat Bilal Saad Jalil, Zahida Hatif Mahdi, Asma Mohammed, Mohamed Omar Mohamed Kaseb This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8636666/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Mainly brought on by Fusarium pseudograminearum , Fusarium crown rot (FCR) is a terrible wheat disease made worse by soil salinity, especially in dry places like Iraq. This research sought to create a dual-strain bio-inoculant including a real halophile and an aggressive lipopeptide producer so as to simultaneously handle abiotic stress and pathogen pressure. From saline soils in Al-Khairat (Karbala) and Al-Kifl (Najaf), respectively, Bacillus amyloliquefaciens strain BA1 and the halophilic Halomonas elongata strain HE1 were recovered. In vitro tests revealed that BA1 showed strong antagonistic action against F. pseudograminearum (85.3% inhibition), which is related to its genetic ability to make lipopeptides (ituA, srfAA). At the same time, the halophilic strain HE1 did well in high salt (6% NaCl) and made a lot more siderophores (90.5%). Molecular characterization showed that while HE1 included the ectoine synthase gene (ectA), confirming its Osmo adaptive strategy, BA1 contained the ACC deaminase gene (acdS) and generated indole-3-acetic acid (IAA). Direct pathogen antagonism by BA1 and abiotic stress resistance by HE1 provide a strong, synergistic approach for plant protection. Offering a good instrument for sustainable agriculture in dry regions, this study is the first to suggest a consortium of B. amyloliquefaciens and H. elongata as a workable, ecologically friendly approach for controlling wheat crown rot under salty conditions. Fusarium pseudograminearum Wheat PGPB Bacillus amyloliquefaciens Halomonas elongata Lipopeptides Halophiles Biological Control Figures Figure 1 Figure 2 1. Introduction Iraq's wheat (Triticum aestivum L.) output is progressively limited by the twin forces of biotic diseases and abiotic stresses. Under ideal conditions, Fusarium pseudograminearum , the causative agent of Fusarium crown rot (FCR), one of the most harmful soil-borne pathogens, can cause yield losses of more than 50%(Li et al., 2022 ). Widespread soil salinity, a common problem in central and southern Iraq that hinders plant development and reduces the effectiveness of traditional chemical controls, greatly worsens FCR(Albdaiwi et al., 2019 ; Karnwal, 2022 ). This complex interaction calls for creative agricultural techniques able to simultaneously reduce pathogen attack and improve plant resistance to osmotic stress. As a sustainable substitute for chemical fertilizers, plant growth-promoting bacteria (PGPB) have been found to improve plant fitness by means of both direct and indirect mechanisms. Mostly as a result of the creation of cyclic lipopeptide medicines (CLPs) including surfactin, iturin, and fengycin, the genus Bacillus is well known for its biocontrol possibilities. These secondary metabolites have a strong antifungal effect against Fusarium species(K. Kim et al., 2017 ; Yuan et al., 2025 ) and can break down the membranes of pathogen cells. Moreover, Bacillus strains frequently generate phytohormones such indole-3-acetic acid (IAA) and the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which control plant ethylene levels and further suppress stress reactions(Ayaz et al., 2022 ; Singh & Jha, 2017 ) . On the other hand, halophilic bacteria like Halomonas elongata have evolved in special ways that let them live happily in places where there is a lot of salt. To keep cellular turgor and metabolic activity, these bacteria use complex osmoadaptation systems including the synthesis of compatible solutes like ectoine (Cánovas et al., 1997 ; Schwibbert et al., 2011 ). Recent studies show that halophiles can act as PGPB, therefore enhancing plant development and nutrient availability in saline soils (Aisha Waheed Qurashi, 2011 ; Kerbab et al., 2021 ). Although individual research on Bacillus spp. and halophiles has been done, the possibility of a combination of a strong lipopeptide producer and a genuine halophile to regulate FCR under salty settings is yet unknown. Under salty circumstances, we speculated that a framework combining a lipopeptide-producing Bacillus amyloliquefaciens with the halophilic Halomonas elongata would provide better protection against FCR in wheat. Therefore, this study seeks to: (i) isolate and identify a lipopeptide-producing Bacillus strain from Iraqi soils as well as a halophilic Halomonas strain; (ii) clarify their unique plant growth-promoting (PGP) and antifungal properties, with a focus on performance under high salinity; and (iii) define the genetic determinants behind their biocontrol and stress-relieving abilities. 2. Materials and Methodology 2.1. Description of the Sample Site and Collection From the rhizosphere soil of two separate sites, (i) Al-Khairat, Karbala (saline-sodic soil) and (ii) Al-Kifl, Najaf (very saline soil), wheat plants were sampled during the bloom stage (March–April 2023). Physicochemical examination of soil samples gave microbial isolation some background information (Table 1). Microbial separation was achieved by aseptic processing of samples. 2.2. Screening and separation of bacteria Soil suspensions were put onto Tryptic Soy Agar (TSA) with 3% NaCl to make it better for halophilic and halotolerant species. Dual culture testing was used to screen a total of 85 bacterial isolates for antagonism against a locally found, pathogenic F. pseudograminearum strain. Comprehensive characterization was decided upon for two most powerful isolates, named BA1 and HE1. 2.3. Molecular Identification and Detection of Functional Genes Strains BA1 and HE1 had genomic DNA isolated from them. Species identification was made by sequencing the 16S rRNA gene amplified with universal primers 27F/1492R. PCR was also used to find important functional genes: for B. amyloliquefaciens BA1, genes for making lipopeptides (srfAA for surfactin and ituA for iturin) and the ACC deaminase gene (acdS) were found using primers that are already known (Cánovas et al., 1997; Schwibbert et al., 2011) for H. elongata HE1, the gene that makes ectoine (ectA) and genes that make siderophores were targeted using primers that are known (Cob-Calan et al., 2019; Sarwar et al., 2018) . 2.4. Definition of Antagonistic and Plant Growth-Promoting Mechanisms Under usual (0% NaCl) and high salinity stress (6% NaCl) settings, all tests were run. The halophilic strain HE1 grows best at a 6% NaCl concentration that also mimics very salty soils. Salkowski's reagent was used to measure the amount of indole-3-acetic acid (IAA) made. Liquid tests and Chrome Azurol S (CAS) agar were used to evaluate siderophore synthesis. Glucose release from a β-glucan substrate was used to ascertain β-glucanase activity. Using a sealed dual-plate test, the antagonistic impact of volatile organic compounds (VOCs) was examined. Three separate biological replicates were used for every experiment (n=3). One-way analysis of variance (ANOVA) was used in SPSS (version 26) to examine data, and Tukey's Honestly Significant Difference (HSD) test at a 95% confidence level (P < 0.05) found significant differences between means. 3. Results 3.1. Molecular identification and isolation of powerful antagonistic strains From rhizosphere soil samples taken in Karbala and Najaf, altogether 85 bacterial isolates were found. Dual culture tests against Fusarium pseudograminearum first screened two isolates, BA1 and HE1, as having the strongest and most consistent antagonistic action. These were picked for thorough characterization. As indicated in Table 1 , the soils of origin had different saline properties. Molecular identification based on 16S rRNA gene sequencing revealed that isolate BA1 shared 99.9% similarity with Bacillus amyloliquefaciens, while isolate HE1 exhibited 99.7% similarity with Halomonas elongata. GenBank accession numbers OR987654 and OR987655 respectively listed sequences deposited there. PCR-based searches for functional genes gave initial indication of their metabolic capacity. H. elongata HE1 had the ectoine synthase gene (ectA), but B. amyloliquefaciens BA1 had genes for lipopeptide synthesis (srfAA, ituA) and the ACC deaminase gene (acdS), as shown in Table 2 . The existence of acdS in BA1 and ectA in HE1 provides a genetic foundation for their predicted functions in osmoadaptation and stress reduction. Table 2 Molecular Identification and Detection of Functional Genes in Bacterial Isolates Isolate Closest Relative (GenBank) Similarity (%) Accession No. Lipopeptide Genes ( srfAA , ituA ) ACC Deaminase Gene ( acdS ) Ectoine Synthase Gene ( ectA ) BA1 Bacillus amyloliquefaciens 99.9 OR987654 + + - HE1 Halomonas elongata 99.7 OR987655 - - + Symbol Legend : + (Present), - (Absent) 3.2. Multidimensional hostile behavior toward Fusarium pseudograminearum Three separate in vitro tests were used to evaluate the antagonistic potential of the two strains in order to understand how the pathogen is suppressed. The data showed a sharp difference in their methods for biological control. Bacillus amyloliquefaciens BA1 proved to be a far better enemy in direct contact situations. Dual culture tests revealed a notable rise over the 65.4% inhibition attained by H. elongata HE1 (Table 3 ), with 85.3% of fungal radial growth inhibited. In the culture filtrate test, metabolites secreted by BA1 showed greater activity against fungus development by 72.1%, whereas HE1 showed only 45.3% (Table 3 ), confirming this better performance. These results clearly back the idea that the effectiveness of BA1 is mostly brought about by the production of strong, dispersible antifungal chemicals, especially the genetically discovered lipopeptides (K. Kim et al., 2017 ; Yuan et al., 2025 ) . Table 3 In Vitro Antifungal Activity of Bacterial Isolates against Fusarium pseudograminearum Bacterial Strain Dual Culture Assay (Inhibition %)* Culture Filtrate Assay (Inhibition %)* VOCs Assay (Inhibition %)* B. amyloliquefaciens BA1 85.3 ± 1.1 a 72.1 ± 1.4 a 61.2 ± 2.1 a H. elongata HE1 65.4 ± 1.6 b 45.3 ± 1.9 b 58.4 ± 1.9 a Control (Fungus only) 0.0 ± 0.0 c 0.0 ± 0.0 c 0.0 ± 0.0 b Values are mean ± SD (n = 3). Different letters within a column indicate significant differences at P < 0.05. Conversely, the difference was less noticeable even if both strains were effective from afar. With no statistically significant difference between them, both BA1 (61.2%) and HE1 (58.4%) showed strong inhibition by the creation of volatile organic compounds (VOCs) (Table 3 , Fig. 1 ). This emphasizes that, regardless of their degree of direct contact, VOC manufacture is a main, common mechanism for both isolates. Figure 1 . Dual culture, culture filtrate, and volatile organic compounds (VOCs) are three distinct methods used in a bar chart to show the percentage inhibition of F. pseudograminearum by B. amyloliquefaciens BA1 and H. elongata HE1. The average percentage inhibition is shown by bars; error bars show the standard deviation (SD) from three biological replicates (n = 3). At P < 0.05, various letters above the bars for every mechanism point to notable differences.3.3. Plant Growth-Promoting Traits and Their Response to Salinity Stress. 3.3. Plant traits that promote growth and their reaction to salinity stress The isolates' plant growth-promoting (PGP) potential was evaluated under normal (0% NaCl) and high salt stress (6% NaCl) settings to determine their functional resilience. Under typical circumstances, both strains displayed encouraging PGP features (Table 4 ). With 48.5 µg/mL of the phytohormone indole-3-acetic acid (IAA), Bacillus amyloliquefaciens BA1 was a prolific producer; this trait is not present in HE1. With BA1 showing somewhat greater activity, both strains generated siderophores and the cell wall-degrading enzyme β-glucanase. Table 4 Plant Growth-Promoting Traits of Bacterial Isolates under Normal Conditions (0% NaCl) Strain IAA Production (µg/mL) Siderophore Production (%) β-Glucanase Activity (U/mL) B. amyloliquefaciens BA1 48.5 ± 1.3 a 60.1 ± 2.2 a 15.8 ± 0.9 a H. elongata HE1 ND 52.3 ± 2.0 b 13.1 ± 0.7 b Control ND 0.0 ± 0.0 c 0.0 ± 0.0 c ND: Not Detected. Values are mean ± SD (n = 3). Different letters within a column indicate significant differences at P < 0.05. Extreme salinity stress response showed clear but complementary adaptive strategies (Table 5 ). BA1 increased its IAA synthesis to 55.1 µg/mL under saline settings, which points to a mechanism to balance salt-induced growth suppression in plants by encouraging root development. On the other hand, HE1, which doesn't make IAA, increased its siderophore output by over 70%, reaching 90.5%. This implies a deliberate change to enhance iron absorption under salt stress to balance nutritional lockout. By illustrating the several possible interactions of objects in Fig. 2 , this varied reaction is made quite apparent: Although BA1 emphasizes hormonal support, HE1 is wonderful at obtaining food under stress. Amazingly, both strains maintained strong β-glucanase activity under salinity even in osmotic stress, but with a slight decrease, hence showing a sustained capacity to dissolve fungus cell walls. Table 5 Plant Growth-Promoting Traits of Bacterial Isolates under Extreme Salinity Stress (6% NaCl) Strain IAA Production (µg/mL) Siderophore Production (%) β-Glucanase Activity (U/mL) B. amyloliquefaciens BA1 55.1 ± 1.5 a 72.4 ± 2.6 a 13.2 ± 0.8 a H. elongata HE1 ND 90.5 ± 3.2 b 11.5 ± 0.6 b Control ND 0.0 ± 0.0 c 0.0 ± 0.0 c ND: Not Detected. Values are mean ± SD (n = 3). Different letters within a column indicate significant differences at P < 0.05. Figure 2 . Bar chart showing how differently the two bacterial strains react to high salt stress (6% NaCl). (A) Illustrates how B. amyloliquefaciens BA1 produces a notably higher level of Indole-3-acetic acid (IAA). (B) Demonstrates H. elongata HE1's spectacular rise in siderophore output. For every panel, bars show the average value; error bars show the standard deviation (SD) from three biological replicates (n = 3). For each strain at P < 0.05, asterisks (*) denote a notable variation between the normal and saline environments. 4. Discussion Our findings show that for creating bio-inoculants meant for difficult environments, a targeted approach is rather effective. Two different types of germs have been found: Halomonas elongata HE1, which acts as the defensive player, and Bacillus amyloliquefaciens BA1, which acts as the offensive player. Considering their combined possibilities, they offer a good strategy to handle soil salinity and Fusarium crown rot, the two primary problems facing wheat. The strong resistance we saw in BA1 is linked to its genes encoding lipopeptides, especially surfactin and iturin. These metabolites are known to cause damage to fungal cell membranes (Y. T. Kim et al., 2020 ; Yuan et al., 2025 ). This simple attack is not the only benefit; BA1 also produces IAA and ACC deaminase. Although IAA helps the plant to better manage stress by lowering the ethylene levels that rise during stress, ACC deaminase boosts root growth (Khan et al., 2017 ; Pourbabaei et al., 2016 ). But even if BA1 is very effective, its performance can decline if salinity gets too high. The most creative feature of this research is how we used the halophile HE1. Its purpose is to specialize in salt, not just to withstand it. Bacteria secreted a lot more siderophores when under salt stress. The existence of the ectoine synthase gene (ectA) supports the conclusion that the bacteria are rather good at adjusting to osmotic stress (Hobmeier et al., 2022 ; Schwibbert et al., 2011 ). By manufacturing ectoine, HE1 regulates its internal cell pressure. Mostly by capturing iron, this helps it to stay alive and support plant development even when other microorganisms cannot live in too harsh conditions (Y. T. Kim et al., 2020 ; Nakayama et al., 2000 ). Since it ensures that biocontrol mechanisms continue functioning precisely when the crop is most vulnerable, this protection plan is especially crucial. Potential Applications and Perspectives Between these lab results and a useable farm product are several important phases. We have to focus on formulation science to make sure both BA1 and HE1 stay alive during storage and after they are used in the field. Carriers such peat, alginate beads, or wheat bran would be good options for this (Albdaiwi et al., 2019 ). Moreover, tests in greenhouses and real-world fields demonstrating that a BA1-HE1 partnership actually performs in the demanding soil conditions are needed. Naturally, these tests should assess grain yield as well as root colonization, indicators of plant health like chlorophyll and water content, and markers for disease control. Long-term improvements in the quality of the soil would likely result from using this bio-inoculant in Iraq together with sustainable farming techniques like rotating legume planting. The Synergistic Theory Given that HE1 and BA1 work together, we believe they would get along better. In salty soils, we believe that combining the two strains will more consistently maintain the illness under check than using either strain alone. While HE1 ensures the therapy remains successful under high salinity by grabbing iron and maintaining its own metabolism working (Khan et al., 2022 ; Yokota & Hayakawa, 2015 ), BA1 serves as the main enemy against the pathogen. The next logical step is to create and test a BA1-HE1 combination in order to confirm this theory and precisely measure the degree of synergy that exists. We expect to see outcomes similar to the synergistic effects seen in other biocontrol studies (Makhlouf et al., 2023 ; Oulmi et al., 2023 ) . 5. Conclusion A strong bacterial combination, the antibiotic-producing Bacillus amyloliquefaciens BA1 and the true halophile Halomonas elongata HE1, was effectively separated and identified in this research. Their unique but complementary ways of working—direct pathogen attack and resistance to abiotic stress—make them a great place to start creating new bioinoculants for the next generation. This study presents a sustainable and very effective way to improve food security in salty and dry areas all around the world by setting up a revolutionary framework for the simultaneous control of salt stress in wheat and Fusarium crown rot. Declarations Author Contributions B.S.J. and Z.H.M. visualize and designed the experiments. A.S.A. performed the experiments. M.O.K. and B.S.J. analysed the data and wrote the manuscript. B.S.J., A.S.A. and Z.H.M. participated in the experimentations and analysis. B.S.J., A.S.A., M.O.K and Z.H.M. edited the manuscript. All authors have read and agreed to the published version of the manuscript. Funding No funding Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The authors declare that supporting data for the study findings are presented in the manuscript, and available from the corresponding author upon request. Conflicts of Interest The authors declare no conflict of interest. References Aisha Waheed Qurashi. (2011). Alleviation of salt stress by Halomonas sp. and osmolytes in Zea mays. AFRICAN JOURNAL OF BIOTECHNOLOGY , 10 (77). https://doi.org/10.5897/AJB11.2084 Albdaiwi, R. N., Khyami-Horani, H., Ayad, J. Y., Alananbeh, K. M., & Al-Sayaydeh, R. (2019). Isolation and Characterization of Halotolerant Plant Growth Promoting Rhizobacteria From Durum Wheat (Triticum turgidum subsp. durum) Cultivated in Saline Areas of the Dead Sea Region. Frontiers in Microbiology , 10 . https://doi.org/10.3389/fmicb.2019.01639 Ayaz, M., Ali, Q., Jiang, Q., Wang, R., Wang, Z., Mu, G., Khan, S. A., Khan, A. R., Manghwar, H., Wu, H., Gao, X., & Gu, Q. (2022). Salt Tolerant Bacillus Strains Improve Plant Growth Traits and Regulation of Phytohormones in Wheat under Salinity Stress. Plants , 11 (20), 2769. https://doi.org/10.3390/plants11202769 Cánovas, D., Vargas, C., Iglesias-Guerra, F., Csonka, L. N., Rhodes, D., Ventosa, A., & Nieto, J. J. (1997). Isolation and Characterization of Salt-sensitive Mutants of the Moderate Halophile Halomonas elongata and Cloning of the Ectoine Synthesis Genes. Journal of Biological Chemistry , 272 (41), 25794–25801. https://doi.org/10.1074/jbc.272.41.25794 Cob-Calan, N. N., Chi-Uluac, L. A., Ortiz-Chi, F., Cerqueda-García, D., Navarrete-Vázquez, G., Ruiz-Sánchez, E., & Hernández-Núñez, E. (2019). Molecular Docking and Dynamics Simulation of Protein β-Tubulin and Antifungal Cyclic Lipopeptides. Molecules , 24 (18), 3387. https://doi.org/10.3390/molecules24183387 Hobmeier, K., Cantone, M., Nguyen, Q. A., Pflüger-Grau, K., Kremling, A., Kunte, H. J., Pfeiffer, F., & Marin-Sanguino, A. (2022). Adaptation to Varying Salinity in Halomonas elongata: Much More Than Ectoine Accumulation. Frontiers in Microbiology , 13 . https://doi.org/10.3389/fmicb.2022.846677 Karnwal, A. (2022). Potential of halotolerant PGPRs in growth and yield augmentation of Triticum aestivum var. HD2687 and Zea mays var. PSCL4642 cultivars under saline conditions. BioTechnologia , 103 (4), 331–342. https://doi.org/10.5114/bta.2022.120703 Kerbab, S., Silini, A., Chenari Bouket, A., Cherif-Silini, H., Eshelli, M., El Houda Rabhi, N., & Belbahri, L. (2021). Mitigation of NaCl Stress in Wheat by Rhizosphere Engineering Using Salt Habitat Adapted PGPR Halotolerant Bacteria. Applied Sciences , 11 (3), 1034. https://doi.org/10.3390/app11031034 Khan, M. Y., Nadeem, S. M., Sohaib, M., Waqas, M. R., Alotaibi, F., Ali, L., Zahir, Z. A., & Al-Barakah, F. N. I. (2022). Potential of plant growth promoting bacterial consortium for improving the growth and yield of wheat under saline conditions. Frontiers in Microbiology , 13 . https://doi.org/10.3389/fmicb.2022.958522 Khan, M. Y., Zahir, Z. A., Asghar, H. N., & Waraich, E. A. (2017). Preliminary investigations on selection of synergistic halotolerant plant growth promoting rhizobacteria for inducing salinity tolerance in wheat. Pakistan Journal of Botany , 49 , 1541–1551. https://api.semanticscholar.org/CorpusID:28617764 Kim, K., Lee, Y., Ha, A., Kim, J.-I., Park, A. R., Yu, N. H., Son, H., Choi, G. J., Park, H. W., Lee, C. W., Lee, T., Lee, Y.-W., & Kim, J.-C. (2017). Chemosensitization of Fusarium graminearum to Chemical Fungicides Using Cyclic Lipopeptides Produced by Bacillus amyloliquefaciens Strain JCK-12. Frontiers in Plant Science , 8 . https://doi.org/10.3389/fpls.2017.02010 Kim, Y. T., Kim, S. E., Lee, W. J., Fumei, Z., Cho, M. S., Moon, J. S., Oh, H.-W., Park, H.-Y., & Kim, S. U. (2020). Isolation and characterization of a high iturin yielding Bacillus velezensis UV mutant with improved antifungal activity. PLOS ONE , 15 (12), e0234177. https://doi.org/10.1371/journal.pone.0234177 Li, S., Xu, J., Fu, L., Xu, G., Lin, X., Qiao, J., & Xia, Y. (2022). Biocontrol of Wheat Crown Rot Using Bacillus halotolerans QTH8. Pathogens , 11 (5), 595. https://doi.org/10.3390/pathogens11050595 Makhlouf, K. E., Boungab, K., & Mokrani, S. (2023). Synergistic effect of Pseudomonas azotoformans and Trichoderma gamsii in management of Fusarium crown rot of wheat. Archives of Phytopathology and Plant Protection , 56 (2), 108–126. https://doi.org/10.1080/03235408.2023.2178056 Nakayama, H., Yoshida, K., Ono, H., Murooka, Y., & Shinmyo, A. (2000). Ectoine, the compatible solute of Halomonas elongata, confers hyperosmotic tolerance in cultured tobacco cells. Plant Physiology , 122 (4), 1239–1248. https://doi.org/10.1104/pp.122.4.1239 Oulmi, A., Bencheikh, A., Mamache, W., Gharzouli, A., Barkahoum Daichi, M., & Rouag, N. (2023). Study of synergistic effect of combined application of tebuconazole with two biocontrol agents for management of Fusarium crown rot in durum wheat. Revista de La Facultad de Agronomía, Universidad Del Zulia , 40 (3), e234025. https://doi.org/10.47280/RevFacAgron(LUZ).v40.n3.03 Pourbabaei, A. A., Bahmani, E., Alikhani, H. A., & Emami, S. (2016). Promotion of Wheat Growth under Salt Stress by Halotolerant Bacteria Containing ACC deaminase. Journal of Agricultural Science and Technology , 18 , 855–864. https://api.semanticscholar.org/CorpusID:3904716 Sarwar, A., Brader, G., Corretto, E., Aleti, G., Abaidullah, M., Sessitsch, A., & Hafeez, F. Y. (2018). Qualitative analysis of biosurfactants from Bacillus species exhibiting antifungal activity. PLOS ONE , 13 (6), e0198107. https://doi.org/10.1371/journal.pone.0198107 Schwibbert, K., Marin‐Sanguino, A., Bagyan, I., Heidrich, G., Lentzen, G., Seitz, H., Rampp, M., Schuster, S. C., Klenk, H., Pfeiffer, F., Oesterhelt, D., & Kunte, H. J. (2011). A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T. Environmental Microbiology , 13 (8), 1973–1994. https://doi.org/10.1111/j.1462-2920.2010.02336.x Singh, R. P., & Jha, P. N. (2017). The PGPR Stenotrophomonas maltophilia SBP-9 Augments Resistance against Biotic and Abiotic Stress in Wheat Plants. Frontiers in Microbiology , 8 . https://doi.org/10.3389/fmicb.2017.01945 Yokota, K., & Hayakawa, H. (2015). Impact of Antimicrobial Lipopeptides from Bacillus sp. on Suppression of Fusarium Yellows of Tatsoi. Microbes and Environments , 30 (3), 281–283. https://doi.org/10.1264/jsme2.ME15062 Yuan, Q., Yang, P., Liu, Y., Tabl, K. M., Guo, M., Zhang, J., Wu, A., Liao, Y., Huang, T., & He, W. (2025). Iturin and fengycin lipopeptides inhibit pathogenic Fusarium by targeting multiple components of the cell membrane and their regulative effects in wheat. Journal of Integrative Plant Biology , 67 (8), 2184–2197. https://doi.org/10.1111/jipb.13933 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 08 May, 2026 Reviewers agreed at journal 08 May, 2026 Reviews received at journal 08 Apr, 2026 Reviewers agreed at journal 01 Apr, 2026 Reviewers agreed at journal 24 Mar, 2026 Reviewers invited by journal 04 Feb, 2026 Editor assigned by journal 04 Feb, 2026 Submission checks completed at journal 23 Jan, 2026 First submitted to journal 19 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8636666","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":586568606,"identity":"da109ff7-46b0-4a0b-8d47-a70b82eae198","order_by":0,"name":"Bilal Saad Jalil","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIie3Qv0rEMBzA8V8I1OWHXXuovVdIKXh3CD5LSsFOt58INaEQn0HwITreGAm0S90LLoqrw7mIbuY8BRHTAyeRfMmSkA/5A+Dz/cEYpQLuAGKAAIAI2F+vRu/DSew2DpB+EtxOYEMy8Y24m+wQueJQFnWrsofT5TGGgl7fIkwPhIPMKlJFHMy87hqT3HQ5RjrIjxCi1EWYIcISPa/7Qo2koggaD/csyQZI9bK+GOuLi1epznGsw+dtRNlTKGf9SUOkMsg0BoPEvkVNOTPJZdfkI9m1mJggnV0x91smoTH9alGOd1uVPMnlWRy31X3/uCidP/Zxva8Tulkhw+SnfkF8Pp/vn/YGNtZTwmtNxtoAAAAASUVORK5CYII=","orcid":"","institution":"University of Kerbala","correspondingAuthor":true,"prefix":"","firstName":"Bilal","middleName":"Saad","lastName":"Jalil","suffix":""},{"id":586568607,"identity":"56274743-acaf-4e49-90bb-1ed0404b6dd7","order_by":1,"name":"Zahida Hatif Mahdi","email":"","orcid":"","institution":"Basic Sciences Department, College of Nursing, University of Kerbala","correspondingAuthor":false,"prefix":"","firstName":"Zahida","middleName":"Hatif","lastName":"Mahdi","suffix":""},{"id":586568608,"identity":"71907960-2cbe-4187-b12a-4edae4957df4","order_by":2,"name":"Asma Mohammed","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Asma","middleName":"","lastName":"Mohammed","suffix":""},{"id":586568609,"identity":"bde29954-f5ce-4836-b18a-cb463c94e761","order_by":3,"name":"Mohamed Omar Mohamed Kaseb","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"Omar Mohamed","lastName":"Kaseb","suffix":""}],"badges":[],"createdAt":"2026-01-19 08:37:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8636666/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8636666/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102040238,"identity":"be612dac-e270-44d7-ba02-bf0da6d36d64","added_by":"auto","created_at":"2026-02-06 12:50:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":76200,"visible":true,"origin":"","legend":"\u003cp\u003eDual culture, culture filtrate, and volatile organic compounds (VOCs) are three distinct methods used in a bar chart to show the percentage inhibition of F. pseudograminearum by B. amyloliquefaciens BA1 and H. elongata HE1. The average percentage inhibition is shown by bars; error bars show the standard deviation (SD) from three biological replicates (n=3). At P \u0026lt; 0.05, various letters above the bars for every mechanism point to notable differences.3.3. Plant Growth-Promoting Traits and Their Response to Salinity Stress\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8636666/v1/1e0ebacb1d72db79dba5cb86.jpg"},{"id":102295422,"identity":"d8d8aec2-2496-4eea-85e7-32ff7c4e50b5","added_by":"auto","created_at":"2026-02-10 10:11:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":69093,"visible":true,"origin":"","legend":"\u003cp\u003eBar chart showing how differently the two bacterial strains react to high salt stress (6% NaCl). (A) Illustrates how B. amyloliquefaciens BA1 produces a notably higher level of Indole-3-acetic acid (IAA). (B) Demonstrates H. elongata HE1's spectacular rise in siderophore output. For every panel, bars show the average value; error bars show the standard deviation (SD) from three biological replicates (n=3). For each strain at P \u0026lt; 0.05, asterisks (*) denote a notable variation between the normal and saline environments.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8636666/v1/63611445efef48582fed4ad1.jpg"},{"id":102298715,"identity":"1269d1b1-9d35-4bce-919e-537236ea1763","added_by":"auto","created_at":"2026-02-10 10:58:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":787554,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8636666/v1/277d266b-f2a4-4051-af83-faa5a5ede40b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functional Characterization of a Novel Bacillus amyloliquefaciens and Halomonas elongata Consortium for Mitigating Fusarium Crown Rot and Salinity Stress in Wheat","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIraq's wheat (Triticum aestivum L.) output is progressively limited by the twin forces of biotic diseases and abiotic stresses. Under ideal conditions, \u003cem\u003eFusarium pseudograminearum\u003c/em\u003e, the causative agent of Fusarium crown rot (FCR), one of the most harmful soil-borne pathogens, can cause yield losses of more than 50%(Li et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Widespread soil salinity, a common problem in central and southern Iraq that hinders plant development and reduces the effectiveness of traditional chemical controls, greatly worsens FCR(Albdaiwi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Karnwal, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This complex interaction calls for creative agricultural techniques able to simultaneously reduce pathogen attack and improve plant resistance to osmotic stress.\u003c/p\u003e \u003cp\u003eAs a sustainable substitute for chemical fertilizers, plant growth-promoting bacteria (PGPB) have been found to improve plant fitness by means of both direct and indirect mechanisms. Mostly as a result of the creation of cyclic lipopeptide medicines (CLPs) including surfactin, iturin, and fengycin, the genus Bacillus is well known for its biocontrol possibilities. These secondary metabolites have a strong antifungal effect against Fusarium species(K. Kim et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yuan et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and can break down the membranes of pathogen cells. Moreover, Bacillus strains frequently generate phytohormones such indole-3-acetic acid (IAA) and the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which control plant ethylene levels and further suppress stress reactions(Ayaz et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Singh \u0026amp; Jha, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) .\u003c/p\u003e \u003cp\u003eOn the other hand, halophilic bacteria like Halomonas elongata have evolved in special ways that let them live happily in places where there is a lot of salt. To keep cellular turgor and metabolic activity, these bacteria use complex osmoadaptation systems including the synthesis of compatible solutes like ectoine (C\u0026aacute;novas et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Schwibbert et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Recent studies show that halophiles can act as PGPB, therefore enhancing plant development and nutrient availability in saline soils (Aisha Waheed Qurashi, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kerbab et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although individual research on Bacillus spp. and halophiles has been done, the possibility of a combination of a strong lipopeptide producer and a genuine halophile to regulate FCR under salty settings is yet unknown.\u003c/p\u003e \u003cp\u003eUnder salty circumstances, we speculated that a framework combining a lipopeptide-producing Bacillus amyloliquefaciens with the halophilic Halomonas elongata would provide better protection against FCR in wheat. Therefore, this study seeks to: (i) isolate and identify a lipopeptide-producing Bacillus strain from Iraqi soils as well as a halophilic Halomonas strain; (ii) clarify their unique plant growth-promoting (PGP) and antifungal properties, with a focus on performance under high salinity; and (iii) define the genetic determinants behind their biocontrol and stress-relieving abilities.\u003c/p\u003e"},{"header":"2. Materials and Methodology","content":"\u003cp\u003e2.1. Description of the Sample Site and Collection From the rhizosphere soil of two separate sites, (i) Al-Khairat, Karbala (saline-sodic soil) and (ii) Al-Kifl, Najaf (very saline soil), wheat plants were sampled during the bloom stage (March\u0026ndash;April 2023). Physicochemical examination of soil samples gave microbial isolation some background information (Table 1). Microbial separation was achieved by aseptic processing of samples.\u003c/p\u003e\n\u003cp\u003e2.2. Screening and separation of bacteria Soil suspensions were put onto Tryptic Soy Agar (TSA) with 3% NaCl to make it better for halophilic and halotolerant species. Dual culture testing was used to screen a total of 85 bacterial isolates for antagonism against a locally found, pathogenic F. pseudograminearum strain. Comprehensive characterization was decided upon for two most powerful isolates, named BA1 and HE1.\u003c/p\u003e\n\u003cp\u003e2.3. Molecular Identification and Detection of Functional Genes Strains BA1 and HE1 had genomic DNA isolated from them. Species identification was made by sequencing the 16S rRNA gene amplified with universal primers 27F/1492R. PCR was also used to find important functional genes: for B. amyloliquefaciens BA1, genes for making lipopeptides (srfAA for surfactin and ituA for iturin) and the ACC deaminase gene (acdS) were found using primers that are already known (C\u0026aacute;novas et al., 1997; Schwibbert et al., 2011) for H. elongata HE1, the gene that makes ectoine (ectA) and genes that make siderophores were targeted using primers that are known (Cob-Calan et al., 2019; Sarwar et al., 2018) .\u003c/p\u003e\n\u003cp\u003e2.4. Definition of Antagonistic and Plant Growth-Promoting Mechanisms Under usual (0% NaCl) and high salinity stress (6% NaCl) settings, all tests were run. The halophilic strain HE1 grows best at a 6% NaCl concentration that also mimics very salty soils. Salkowski\u0026apos;s reagent was used to measure the amount of indole-3-acetic acid (IAA) made. Liquid tests and Chrome Azurol S (CAS) agar were used to evaluate siderophore synthesis. Glucose release from a \u0026beta;-glucan substrate was used to ascertain \u0026beta;-glucanase activity. Using a sealed dual-plate test, the antagonistic impact of volatile organic compounds (VOCs) was examined. Three separate biological replicates were used for every experiment (n=3). One-way analysis of variance (ANOVA) was used in SPSS (version 26) to examine data, and Tukey\u0026apos;s Honestly Significant Difference (HSD) test at a 95% confidence level (P \u0026lt; 0.05) found significant differences between means.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1. Molecular identification and isolation of powerful antagonistic strains From rhizosphere soil samples taken in Karbala and Najaf, altogether 85 bacterial isolates were found. Dual culture tests against Fusarium pseudograminearum first screened two isolates, BA1 and HE1, as having the strongest and most consistent antagonistic action. These were picked for thorough characterization. As indicated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the soils of origin had different saline properties.\u003c/p\u003e \u003cp\u003eMolecular identification based on 16S rRNA gene sequencing revealed that isolate BA1 shared 99.9% similarity with Bacillus amyloliquefaciens, while isolate HE1 exhibited 99.7% similarity with Halomonas elongata. GenBank accession numbers OR987654 and OR987655 respectively listed sequences deposited there. PCR-based searches for functional genes gave initial indication of their metabolic capacity. H. elongata HE1 had the ectoine synthase gene (ectA), but B. amyloliquefaciens BA1 had genes for lipopeptide synthesis (srfAA, ituA) and the ACC deaminase gene (acdS), as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The existence of acdS in BA1 and ectA in HE1 provides a genetic foundation for their predicted functions in osmoadaptation and stress reduction.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMolecular Identification and Detection of Functional Genes in Bacterial Isolates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClosest Relative (GenBank)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSimilarity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAccession No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLipopeptide Genes (\u003cem\u003esrfAA\u003c/em\u003e, \u003cem\u003eituA\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eACC Deaminase Gene (\u003cem\u003eacdS\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEctoine Synthase Gene (\u003cem\u003eectA\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOR987654\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHE1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHalomonas elongata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOR987655\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSymbol Legend\u003c/em\u003e:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ (Present), - (Absent)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e3.2. Multidimensional hostile behavior toward Fusarium pseudograminearum Three separate in vitro tests were used to evaluate the antagonistic potential of the two strains in order to understand how the pathogen is suppressed. The data showed a sharp difference in their methods for biological control. Bacillus amyloliquefaciens BA1 proved to be a far better enemy in direct contact situations. Dual culture tests revealed a notable rise over the 65.4% inhibition attained by H. elongata HE1 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), with 85.3% of fungal radial growth inhibited. In the culture filtrate test, metabolites secreted by BA1 showed greater activity against fungus development by 72.1%, whereas HE1 showed only 45.3% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), confirming this better performance. These results clearly back the idea that the effectiveness of BA1 is mostly brought about by the production of strong, dispersible antifungal chemicals, especially the genetically discovered lipopeptides (K. Kim et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yuan et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) .\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIn Vitro Antifungal Activity of Bacterial Isolates against \u003cem\u003eFusarium pseudograminearum\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBacterial Strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDual Culture Assay (Inhibition %)*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCulture Filtrate Assay (Inhibition %)*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVOCs Assay (Inhibition %)*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e BA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. elongata\u003c/em\u003e HE1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl (Fungus only)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cem\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). Different letters within a column indicate significant differences at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eConversely, the difference was less noticeable even if both strains were effective from afar. With no statistically significant difference between them, both BA1 (61.2%) and HE1 (58.4%) showed strong inhibition by the creation of volatile organic compounds (VOCs) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This emphasizes that, regardless of their degree of direct contact, VOC manufacture is a main, common mechanism for both isolates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Dual culture, culture filtrate, and volatile organic compounds (VOCs) are three distinct methods used in a bar chart to show the percentage inhibition of F. pseudograminearum by B. amyloliquefaciens BA1 and H. elongata HE1. The average percentage inhibition is shown by bars; error bars show the standard deviation (SD) from three biological replicates (n\u0026thinsp;=\u0026thinsp;3). At P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, various letters above the bars for every mechanism point to notable differences.3.3. Plant Growth-Promoting Traits and Their Response to Salinity Stress.\u003c/p\u003e \u003cp\u003e3.3. Plant traits that promote growth and their reaction to salinity stress The isolates' plant growth-promoting (PGP) potential was evaluated under normal (0% NaCl) and high salt stress (6% NaCl) settings to determine their functional resilience.\u003c/p\u003e \u003cp\u003eUnder typical circumstances, both strains displayed encouraging PGP features (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). With 48.5 \u0026micro;g/mL of the phytohormone indole-3-acetic acid (IAA), Bacillus amyloliquefaciens BA1 was a prolific producer; this trait is not present in HE1. With BA1 showing somewhat greater activity, both strains generated siderophores and the cell wall-degrading enzyme β-glucanase.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePlant Growth-Promoting Traits of Bacterial Isolates under Normal Conditions (0% NaCl)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIAA Production (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSiderophore Production (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ-Glucanase Activity (U/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e BA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. elongata\u003c/em\u003e HE1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cem\u003eND: Not Detected. Values are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). Different letters within a column indicate significant differences at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eExtreme salinity stress response showed clear but complementary adaptive strategies (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). BA1 increased its IAA synthesis to 55.1 \u0026micro;g/mL under saline settings, which points to a mechanism to balance salt-induced growth suppression in plants by encouraging root development. On the other hand, HE1, which doesn't make IAA, increased its siderophore output by over 70%, reaching 90.5%. This implies a deliberate change to enhance iron absorption under salt stress to balance nutritional lockout. By illustrating the several possible interactions of objects in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, this varied reaction is made quite apparent: Although BA1 emphasizes hormonal support, HE1 is wonderful at obtaining food under stress. Amazingly, both strains maintained strong β-glucanase activity under salinity even in osmotic stress, but with a slight decrease, hence showing a sustained capacity to dissolve fungus cell walls.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePlant Growth-Promoting Traits of Bacterial Isolates under Extreme Salinity Stress (6% NaCl)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIAA Production (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSiderophore Production (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ-Glucanase Activity (U/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e BA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. elongata\u003c/em\u003e HE1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cem\u003eND: Not Detected. Values are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). Different letters within a column indicate significant differences at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Bar chart showing how differently the two bacterial strains react to high salt stress (6% NaCl). (A) Illustrates how B. amyloliquefaciens BA1 produces a notably higher level of Indole-3-acetic acid (IAA). (B) Demonstrates H. elongata HE1's spectacular rise in siderophore output. For every panel, bars show the average value; error bars show the standard deviation (SD) from three biological replicates (n\u0026thinsp;=\u0026thinsp;3). For each strain at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, asterisks (*) denote a notable variation between the normal and saline environments.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur findings show that for creating bio-inoculants meant for difficult environments, a targeted approach is rather effective. Two different types of germs have been found: Halomonas elongata HE1, which acts as the defensive player, and Bacillus amyloliquefaciens BA1, which acts as the offensive player. Considering their combined possibilities, they offer a good strategy to handle soil salinity and Fusarium crown rot, the two primary problems facing wheat.\u003c/p\u003e \u003cp\u003eThe strong resistance we saw in BA1 is linked to its genes encoding lipopeptides, especially surfactin and iturin. These metabolites are known to cause damage to fungal cell membranes (Y. T. Kim et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yuan et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This simple attack is not the only benefit; BA1 also produces IAA and ACC deaminase. Although IAA helps the plant to better manage stress by lowering the ethylene levels that rise during stress, ACC deaminase boosts root growth (Khan et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pourbabaei et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). But even if BA1 is very effective, its performance can decline if salinity gets too high.\u003c/p\u003e \u003cp\u003eThe most creative feature of this research is how we used the halophile HE1. Its purpose is to specialize in salt, not just to withstand it. Bacteria secreted a lot more siderophores when under salt stress. The existence of the ectoine synthase gene (ectA) supports the conclusion that the bacteria are rather good at adjusting to osmotic stress (Hobmeier et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Schwibbert et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). By manufacturing ectoine, HE1 regulates its internal cell pressure. Mostly by capturing iron, this helps it to stay alive and support plant development even when other microorganisms cannot live in too harsh conditions (Y. T. Kim et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nakayama et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Since it ensures that biocontrol mechanisms continue functioning precisely when the crop is most vulnerable, this protection plan is especially crucial.\u003c/p\u003e \u003cp\u003ePotential Applications and Perspectives\u003c/p\u003e \u003cp\u003eBetween these lab results and a useable farm product are several important phases. We have to focus on formulation science to make sure both BA1 and HE1 stay alive during storage and after they are used in the field. Carriers such peat, alginate beads, or wheat bran would be good options for this (Albdaiwi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, tests in greenhouses and real-world fields demonstrating that a BA1-HE1 partnership actually performs in the demanding soil conditions are needed. Naturally, these tests should assess grain yield as well as root colonization, indicators of plant health like chlorophyll and water content, and markers for disease control. Long-term improvements in the quality of the soil would likely result from using this bio-inoculant in Iraq together with sustainable farming techniques like rotating legume planting.\u003c/p\u003e \u003cp\u003eThe Synergistic Theory\u003c/p\u003e \u003cp\u003eGiven that HE1 and BA1 work together, we believe they would get along better. In salty soils, we believe that combining the two strains will more consistently maintain the illness under check than using either strain alone. While HE1 ensures the therapy remains successful under high salinity by grabbing iron and maintaining its own metabolism working (Khan et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yokota \u0026amp; Hayakawa, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), BA1 serves as the main enemy against the pathogen. The next logical step is to create and test a BA1-HE1 combination in order to confirm this theory and precisely measure the degree of synergy that exists. We expect to see outcomes similar to the synergistic effects seen in other biocontrol studies (Makhlouf et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Oulmi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) .\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eA strong bacterial combination, the antibiotic-producing Bacillus amyloliquefaciens BA1 and the true halophile Halomonas elongata HE1, was effectively separated and identified in this research. Their unique but complementary ways of working\u0026mdash;direct pathogen attack and resistance to abiotic stress\u0026mdash;make them a great place to start creating new bioinoculants for the next generation. This study presents a sustainable and very effective way to improve food security in salty and dry areas all around the world by setting up a revolutionary framework for the simultaneous control of salt stress in wheat and Fusarium crown rot.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB.S.J. and Z.H.M. visualize and designed the experiments. A.S.A. performed the experiments. M.O.K. and B.S.J. analysed the data and wrote the manuscript. B.S.J., A.S.A. \u0026nbsp;and Z.H.M. participated in the experimentations and analysis. B.S.J., A.S.A., M.O.K and Z.H.M. edited the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that supporting data for the study findings are presented in the manuscript, and available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAisha Waheed Qurashi. (2011). Alleviation of salt stress by Halomonas sp. and osmolytes in Zea mays. \u003cem\u003eAFRICAN JOURNAL OF BIOTECHNOLOGY\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(77). https://doi.org/10.5897/AJB11.2084\u003c/li\u003e\n\u003cli\u003eAlbdaiwi, R. N., Khyami-Horani, H., Ayad, J. Y., Alananbeh, K. M., \u0026amp; Al-Sayaydeh, R. (2019). Isolation and Characterization of Halotolerant Plant Growth Promoting Rhizobacteria From Durum Wheat (Triticum turgidum subsp. durum) Cultivated in Saline Areas of the Dead Sea Region. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e. https://doi.org/10.3389/fmicb.2019.01639\u003c/li\u003e\n\u003cli\u003eAyaz, M., Ali, Q., Jiang, Q., Wang, R., Wang, Z., Mu, G., Khan, S. A., Khan, A. R., Manghwar, H., Wu, H., Gao, X., \u0026amp; Gu, Q. (2022). Salt Tolerant Bacillus Strains Improve Plant Growth Traits and Regulation of Phytohormones in Wheat under Salinity Stress. \u003cem\u003ePlants\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(20), 2769. https://doi.org/10.3390/plants11202769\u003c/li\u003e\n\u003cli\u003eC\u0026aacute;novas, D., Vargas, C., Iglesias-Guerra, F., Csonka, L. N., Rhodes, D., Ventosa, A., \u0026amp; Nieto, J. J. (1997). Isolation and Characterization of Salt-sensitive Mutants of the Moderate Halophile Halomonas elongata and Cloning of the Ectoine Synthesis Genes. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e, \u003cem\u003e272\u003c/em\u003e(41), 25794\u0026ndash;25801. https://doi.org/10.1074/jbc.272.41.25794\u003c/li\u003e\n\u003cli\u003eCob-Calan, N. N., Chi-Uluac, L. A., Ortiz-Chi, F., Cerqueda-Garc\u0026iacute;a, D., Navarrete-V\u0026aacute;zquez, G., Ruiz-S\u0026aacute;nchez, E., \u0026amp; Hern\u0026aacute;ndez-N\u0026uacute;\u0026ntilde;ez, E. (2019). Molecular Docking and Dynamics Simulation of Protein \u0026beta;-Tubulin and Antifungal Cyclic Lipopeptides. \u003cem\u003eMolecules\u003c/em\u003e, \u003cem\u003e24\u003c/em\u003e(18), 3387. https://doi.org/10.3390/molecules24183387\u003c/li\u003e\n\u003cli\u003eHobmeier, K., Cantone, M., Nguyen, Q. A., Pfl\u0026uuml;ger-Grau, K., Kremling, A., Kunte, H. J., Pfeiffer, F., \u0026amp; Marin-Sanguino, A. (2022). Adaptation to Varying Salinity in Halomonas elongata: Much More Than Ectoine Accumulation. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e. https://doi.org/10.3389/fmicb.2022.846677\u003c/li\u003e\n\u003cli\u003eKarnwal, A. (2022). Potential of halotolerant PGPRs in growth and yield augmentation of Triticum aestivum var. HD2687 and Zea mays var. PSCL4642 cultivars under saline conditions. \u003cem\u003eBioTechnologia\u003c/em\u003e, \u003cem\u003e103\u003c/em\u003e(4), 331\u0026ndash;342. https://doi.org/10.5114/bta.2022.120703\u003c/li\u003e\n\u003cli\u003eKerbab, S., Silini, A., Chenari Bouket, A., Cherif-Silini, H., Eshelli, M., El Houda Rabhi, N., \u0026amp; Belbahri, L. (2021). Mitigation of NaCl Stress in Wheat by Rhizosphere Engineering Using Salt Habitat Adapted PGPR Halotolerant Bacteria. \u003cem\u003eApplied Sciences\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(3), 1034. https://doi.org/10.3390/app11031034\u003c/li\u003e\n\u003cli\u003eKhan, M. Y., Nadeem, S. M., Sohaib, M., Waqas, M. R., Alotaibi, F., Ali, L., Zahir, Z. A., \u0026amp; Al-Barakah, F. N. I. (2022). Potential of plant growth promoting bacterial consortium for improving the growth and yield of wheat under saline conditions. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e. https://doi.org/10.3389/fmicb.2022.958522\u003c/li\u003e\n\u003cli\u003eKhan, M. Y., Zahir, Z. A., Asghar, H. N., \u0026amp; Waraich, E. A. (2017). Preliminary investigations on selection of synergistic halotolerant plant growth promoting rhizobacteria for inducing salinity tolerance in wheat. \u003cem\u003ePakistan Journal of Botany\u003c/em\u003e, \u003cem\u003e49\u003c/em\u003e, 1541\u0026ndash;1551. https://api.semanticscholar.org/CorpusID:28617764\u003c/li\u003e\n\u003cli\u003eKim, K., Lee, Y., Ha, A., Kim, J.-I., Park, A. R., Yu, N. H., Son, H., Choi, G. J., Park, H. W., Lee, C. W., Lee, T., Lee, Y.-W., \u0026amp; Kim, J.-C. (2017). Chemosensitization of Fusarium graminearum to Chemical Fungicides Using Cyclic Lipopeptides Produced by Bacillus amyloliquefaciens Strain JCK-12. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e. https://doi.org/10.3389/fpls.2017.02010\u003c/li\u003e\n\u003cli\u003eKim, Y. T., Kim, S. E., Lee, W. J., Fumei, Z., Cho, M. S., Moon, J. S., Oh, H.-W., Park, H.-Y., \u0026amp; Kim, S. U. (2020). Isolation and characterization of a high iturin yielding Bacillus velezensis UV mutant with improved antifungal activity. \u003cem\u003ePLOS ONE\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(12), e0234177. https://doi.org/10.1371/journal.pone.0234177\u003c/li\u003e\n\u003cli\u003eLi, S., Xu, J., Fu, L., Xu, G., Lin, X., Qiao, J., \u0026amp; Xia, Y. (2022). Biocontrol of Wheat Crown Rot Using Bacillus halotolerans QTH8. \u003cem\u003ePathogens\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(5), 595. https://doi.org/10.3390/pathogens11050595\u003c/li\u003e\n\u003cli\u003eMakhlouf, K. E., Boungab, K., \u0026amp; Mokrani, S. (2023). Synergistic effect of Pseudomonas azotoformans and Trichoderma gamsii in management of Fusarium crown rot of wheat. \u003cem\u003eArchives of Phytopathology and Plant Protection\u003c/em\u003e, \u003cem\u003e56\u003c/em\u003e(2), 108\u0026ndash;126. https://doi.org/10.1080/03235408.2023.2178056\u003c/li\u003e\n\u003cli\u003eNakayama, H., Yoshida, K., Ono, H., Murooka, Y., \u0026amp; Shinmyo, A. (2000). Ectoine, the compatible solute of Halomonas elongata, confers hyperosmotic tolerance in cultured tobacco cells. \u003cem\u003ePlant Physiology\u003c/em\u003e, \u003cem\u003e122\u003c/em\u003e(4), 1239\u0026ndash;1248. https://doi.org/10.1104/pp.122.4.1239\u003c/li\u003e\n\u003cli\u003eOulmi, A., Bencheikh, A., Mamache, W., Gharzouli, A., Barkahoum Daichi, M., \u0026amp; Rouag, N. (2023). Study of synergistic effect of combined application of tebuconazole with two biocontrol agents for management of Fusarium crown rot in durum wheat. \u003cem\u003eRevista de La Facultad de Agronom\u0026iacute;a, Universidad Del Zulia\u003c/em\u003e, \u003cem\u003e40\u003c/em\u003e(3), e234025. https://doi.org/10.47280/RevFacAgron(LUZ).v40.n3.03\u003c/li\u003e\n\u003cli\u003ePourbabaei, A. A., Bahmani, E., Alikhani, H. A., \u0026amp; Emami, S. (2016). Promotion of Wheat Growth under Salt Stress by Halotolerant Bacteria Containing ACC deaminase. \u003cem\u003eJournal of Agricultural Science and Technology\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e, 855\u0026ndash;864. https://api.semanticscholar.org/CorpusID:3904716\u003c/li\u003e\n\u003cli\u003eSarwar, A., Brader, G., Corretto, E., Aleti, G., Abaidullah, M., Sessitsch, A., \u0026amp; Hafeez, F. Y. (2018). Qualitative analysis of biosurfactants from Bacillus species exhibiting antifungal activity. \u003cem\u003ePLOS ONE\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(6), e0198107. https://doi.org/10.1371/journal.pone.0198107\u003c/li\u003e\n\u003cli\u003eSchwibbert, K., Marin‐Sanguino, A., Bagyan, I., Heidrich, G., Lentzen, G., Seitz, H., Rampp, M., Schuster, S. C., Klenk, H., Pfeiffer, F., Oesterhelt, D., \u0026amp; Kunte, H. J. (2011). A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T. \u003cem\u003eEnvironmental Microbiology\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(8), 1973\u0026ndash;1994. https://doi.org/10.1111/j.1462-2920.2010.02336.x\u003c/li\u003e\n\u003cli\u003eSingh, R. P., \u0026amp; Jha, P. N. (2017). The PGPR Stenotrophomonas maltophilia SBP-9 Augments Resistance against Biotic and Abiotic Stress in Wheat Plants. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e. https://doi.org/10.3389/fmicb.2017.01945\u003c/li\u003e\n\u003cli\u003eYokota, K., \u0026amp; Hayakawa, H. (2015). Impact of Antimicrobial Lipopeptides from Bacillus sp. on Suppression of Fusarium Yellows of Tatsoi. \u003cem\u003eMicrobes and Environments\u003c/em\u003e, \u003cem\u003e30\u003c/em\u003e(3), 281\u0026ndash;283. https://doi.org/10.1264/jsme2.ME15062\u003c/li\u003e\n\u003cli\u003eYuan, Q., Yang, P., Liu, Y., Tabl, K. M., Guo, M., Zhang, J., Wu, A., Liao, Y., Huang, T., \u0026amp; He, W. (2025). Iturin and fengycin lipopeptides inhibit pathogenic Fusarium by targeting multiple components of the cell membrane and their regulative effects in wheat. \u003cem\u003eJournal of Integrative Plant Biology\u003c/em\u003e, \u003cem\u003e67\u003c/em\u003e(8), 2184\u0026ndash;2197. https://doi.org/10.1111/jipb.13933\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Fusarium pseudograminearum, Wheat, PGPB, Bacillus amyloliquefaciens, Halomonas elongata, Lipopeptides, Halophiles, Biological Control","lastPublishedDoi":"10.21203/rs.3.rs-8636666/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8636666/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMainly brought on by \u003cem\u003eFusarium pseudograminearum\u003c/em\u003e, Fusarium crown rot (FCR) is a terrible wheat disease made worse by soil salinity, especially in dry places like Iraq. This research sought to create a dual-strain bio-inoculant including a real halophile and an aggressive lipopeptide producer so as to simultaneously handle abiotic stress and pathogen pressure. From saline soils in Al-Khairat (Karbala) and Al-Kifl (Najaf), respectively, \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e strain BA1 and the halophilic Halomonas elongata strain HE1 were recovered. In vitro tests revealed that BA1 showed strong antagonistic action against \u003cem\u003eF. pseudograminearum\u003c/em\u003e (85.3% inhibition), which is related to its genetic ability to make lipopeptides (ituA, srfAA). At the same time, the halophilic strain HE1 did well in high salt (6% NaCl) and made a lot more siderophores (90.5%). Molecular characterization showed that while HE1 included the ectoine synthase gene (ectA), confirming its Osmo adaptive strategy, BA1 contained the ACC deaminase gene (acdS) and generated indole-3-acetic acid (IAA). Direct pathogen antagonism by BA1 and abiotic stress resistance by HE1 provide a strong, synergistic approach for plant protection. Offering a good instrument for sustainable agriculture in dry regions, this study is the first to suggest a consortium of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e and H. elongata as a workable, ecologically friendly approach for controlling wheat crown rot under salty conditions.\u003c/p\u003e","manuscriptTitle":"Functional Characterization of a Novel Bacillus amyloliquefaciens and Halomonas elongata Consortium for Mitigating Fusarium Crown Rot and Salinity Stress in Wheat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-06 12:50:27","doi":"10.21203/rs.3.rs-8636666/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"48942272328960774381973327515206628044","date":"2026-05-08T15:00:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"19594459526044190222880211845077590041","date":"2026-05-08T14:52:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T02:17:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324030639939615568373044001191374856604","date":"2026-04-02T02:13:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121150593002711696228505329901211134493","date":"2026-03-24T17:13:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-04T10:11:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-04T10:06:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-23T10:58:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Microbiology","date":"2026-01-19T07:14:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0f95ea70-c357-47bc-95c6-3336d5fd1b33","owner":[],"postedDate":"February 6th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"48942272328960774381973327515206628044","date":"2026-05-08T15:00:45+00:00","index":108,"fulltext":""},{"type":"reviewerAgreed","content":"19594459526044190222880211845077590041","date":"2026-05-08T14:52:16+00:00","index":106,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-06T12:50:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-06 12:50:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8636666","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8636666","identity":"rs-8636666","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}