Acta agriculturae Slovenica, 120/4, 1–11, Ljubljana 2024 doi:10.14720/aas.2024.120.4.19524 Original research article / izvirni znanstveni članek Utilization of nitrogen-fixing endophytic bacteria to improve tuber yield and nitrogenous nutrient uptake of Cassava plant (Manihot esculenta Crantz) Quang Trung DO 1, 2, Manh Ha NGUYEN 3 Received August 13, 2024; accepted October 26, 2024 Delo je prispelo 13 avgust 2024, sprejeto 26. oktober 2024 1 Faculty of Pharmacy, Dai Nam University, Xom, Phu Lam, Ha Dong, Ha Noi, Vietnam 2 Corresponding author: trungcnsinh@gmail.com 3 Forest Protection Research Center, Vietnamese Academy of Forest Sciences, Duc Thang, Bac Tu Liem, Hanoi, Vietnam Utilization of nitrogen-fixing endophytic bacteria to improve tuber yield and nitrogenous nutrient uptake of Cassava plant (Manihot esculenta Crantz) Abstract: Cassava (Manihot esculenta Cratz) yield remains low due to various environmental stressors, yet sustainable strategies for enhancing productivity, especially through mi- crobial interventions, are underexplored in current literature. This study investigated the potential of endophytic bacteria iso- lated from native cassava (M. esculenta KM98-7) to enhance plant growth and yield under greenhouse and field conditions. Eleven bacterial strains (TL1 to TL11) were isolated and as- sessed for nitrogen fixation, phosphate solubilization, and in- dole acetic acid (IAA) synthesis. TL4 and TL8 exhibited supe- rior capabilities, with TL8 producing the highest levels of NH4+ (14.95 mg l-1) and IAA (46.57 mg l-1). Molecular identification revealed that TL4 and TL8 were closely related to Burkhold- eria cenocepacia Vandamme et al. 2003 and Prestia aryabhattai (Shivaji et al. 2009) Gupta et al. 2020. Greenhouse trials showed that inoculation with TL8 significantly increased plant height, leaf number, and tuber yield, comparable to 90 kg urea ha-1 ap- plication. Field experiments confirmed these findings, with the 60 kg urea ha-1 + TL8 treatment achieving similar yields to 90 kg urea ha-1 without bacterial inoculation. This study demon- strates that integrating nitrogen-fixing bacterial inoculants, particularly strain TL8, with reduced nitrogen fertilization can maintain high cassava productivity while promoting sustain- able agricultural practices. Key words: biofertilizers, crop productivity, endophytic bacteria, plant-growth promotion, sustainable agriculture Uporaba endofitskih bakterij, ki vežejo dušik za izboljša- nje pridelka in privzema hranil manioke (Manihot esculenta Crantz) Izvleček: Pridelek manioke (Manihot esculenta Cratz) ostaja majhen zaradi različnih okoljskih stresnih dejavnikov a kljub obstoječim trajnostnim strategijam za povečanje pridelka, še posebej z mikrobi, ostajajo te neuporabljene glede na obstoječe vire. V raziskavi je bil preučevan potencial end- ofitskih bakterij izoliranih iz lokalne manioke (M. esculenta KM98-7) na vzpodbujanje rasti in povečanje pridelka v ras- tlinjaku in poljskih razmerah. Enajst sevov bakterij (TL1 do TL11) je bilo izoliranih in ocenjenih na vezavo dušika, razta- planje fosfata, in sintezo indol acetne kisline (IAA). Seva TL4 in TL8 sta pokazala najboljšo sposobnost, pri čemer je sev TL8 proizvedel največ NH4+ (14,95 mg l-1) in IAA (46,57 mg l-1). Molekularno preverjanje je odkrilo, da sta bila seva TL4 in TL8 zelo sorodna vrstama Burkholderia cenocepacia Van- damme et al. 2003 in Prestia aryabhattai (Shivaji et al. 2009) Gupta et al. 2020. Poskus v rastlinajku je pokazal, da je inoku- lacija s sevom TL8 značilno povečala višino rastlin, število listov in pridelek gomoljev, ki je bil primerljiv uporabi 90 kg urea ha-1. Poljski poskus je potrdil ta odkritja, kjer je obravna- vanje s 60 kg urea ha-1 + TL8 doseglo podoben pridelek kot uporaba 90 kg urea ha-1 brez bakterijske inokulacije. Raziska- va kaže, da vključevanje inokulov bakterij, ki vežejo dušik, še posebej seva TL8, z zmanjšanjem gnojenja z dušikovimi gno- jili, lahko ohranja velike pridelke manioke in hkrati pospešuje trajnostno kmetijsko pridelavo. Ključne besede: biognojila, pridelek gojenih rastlin, endofitke bakterije, pospeševanje rasti rastlin, trajnostno kmetijstvo Acta agriculturae Slovenica, 120/4 – 20242 Q. T. DO et al. 1 NTRODUCTION Cassava (Manihot esculenta Crantz) is a crucial staple food crop in tropical regions, particularly in sub-Saharan Africa, Latin America, and Southeast Asia, providing a primary source of carbohydrates for millions of people. However, achieving high yields, such as 30 tons of cassava tubers per hectare, requires substantial nutrient removal from the soil - approxi- mately 180-200 kg N ha-1, 15-22 kg P2O5 ha -1, and 140- 160 kg K2O ha -1 (Susan et al., 2010). Consequently, cassava productivity is often constrained by poor soil conditions, such as alum soil, characterized by high acidity and low nutrient availability (Omondi et al., 2018). Traditional agricultural practices aimed at im- proving soil fertility frequently fall short, necessitating alternative, sustainable solutions to enhance cassava growth and yield. One promising approach is the use of plant en- dophytic bacteria, which inhabit plant tissues with- out causing harm and can confer various benefits to their host plants. These bacteria promote plant growth through mechanisms such as nitrogen fixation, phos- phate solubilization, production of growth hormones, and induction of systemic resistance to pathogens (Ferreira et al., 2021; Do et al., 2023; Ferreira et al., 2024). The potential of these bacteria to improve plant health and productivity under stress conditions has been increasingly recognized (Do et al., 2023). Recent studies have highlighted the positive im- pacts of endophytic bacterial inoculation on various crops under different stress conditions. For instance, inoculation with Bacillus sp. has been shown to en- hance the growth and yield of rice in the presence of the bacterium Xanthomonas oryzae pv. Oryzae (Xoo) that caused bacterial leaf blight disease (Do et al., 2023). Similarly, Pseudomonas fluorescens (Flüg- ge 1886) Migula, 1895 strains have been reported to improve the growth of wheat under drought stress by enhancing root development and water uptake (Vuru- konda et al., 2016). These findings suggest that endo- phytic bacteria could be a valuable tool in mitigating abiotic stresses and improving crop productivity on challenging soils. In the context of cassava, research on the role of endophytic bacteria is still emerging. Recent studies (Ferreira et al., 2021; Feng et al., 2023; Ferreira et al., 2024) demonstrated that endophytic bacteria isolated from cassava plants could promote plant growth and enhance resistance against phytopathogens such as Phytopythium sp., a causal agent of soft root rot in cas- sava, and Xanthomonas phaseoli pv. Manihotis (Xpm) which causes cassava bacterial blight. Furthermore, the study by Zhang et al., (2022) indicated that cassa- va-associated endophytes could improve nutrient up- take, leading to better growth and higher yields. Given these promising findings, the present study aims to investigate the effect of plant endophytic bac- terial inoculation on cassava growth and yield. By un- derstanding how these bacteria interact with cassava plants and influence their growth under adverse soil conditions, we can develop sustainable agricultural practices that leverage natural microbial relationships to enhance crop productivity and resilience. 2 MATERIALS AND METHODS 2.1 ISOLATION OF ENDOPHYTIC BACTERIA FROM THE CASSAVA PLANT A total of 50 cassava (Manihot esculenta KM98- 7) plant samples, including roots, stems, and leaves, were collected from Tuy Lai commune in My Duc dis- trict, Ha Noi, Vietnam. These samples were cleaned and cut into 1 cm pieces. Surface sterilization involved immersion in 70 % ethanol with gentle shaking for 3 minutes, followed by rinsing with sterile distilled wa- ter. The samples were then treated with 0.1  % HgCl2 for 1 minute with shaking, rinsed again, treated with 3 % hydrogen peroxide for 3 minutes, and thoroughly rinsed with sterile distilled water four times. To ensure no residual microorganisms, 100 µl of the final rinse water was plated onto Luria-Bertani (LB) medium. Successful sterilization was confirmed by the absence of colonies after 24-48 hours of incubation. The sterilized samples were ground using a sterile mortar and pestle with 1 ml of sterile distilled water. A 100 µl aliquot of the homogenized sample was in- oculated into test tubes containing 3 ml of semi-solid LB medium and incubated at 30  °C for 24-48 hours. The presence of endophytic microorganisms was indicated by a thin film on the medium surface, which was transferred to a solid LB medium and incubated at 30 °C for 24-48 hours. Pure cultures were obtained through subculturing and preserved on slant LB agar tubes at 4 °C. 2.2 CHARACTERIZATION OF ENDOPHYTIC BACTERIA FROM THE CASSAVA PLANT 2.2.1 Nitrogen fixation activity assay All bacterial strains were screened for nitrogen fixa- Acta agriculturae Slovenica, 120/4 – 2024 3 Utilization of nitrogen-fixing endophytic bacteria to ... yield and nitrogenous nutrient uptake of Cassava plant (Manihot esculenta Crantz) tion abilities by culturing in 50 ml nitrogen-free Burk’s liquid medium at room temperature for 3 days with shaking at 160 rpm, followed by centrifugation at 10,000 rpm for 10 minutes. The NH4 + content in the supernatant was quantified using Nessler’s reagent colorimetric meth- od at 420 nm with NH4Cl as the standard (Franche et al., 2009). Specifically, 0.1 ml of sodium potassium tartrate solution and 0.1 ml of Nessler’s reagent were added to 5 ml of the supernatant. After mixing and allowing it to sit for 20 minutes, the absorbance was recorded at 420 nm. A calibration curve for concentration versus absorbance was created using a series of standard NH4Cl solutions with varying concentrations. The resulting curve dem- onstrated a strong linear correlation between absorbance and NH3 concentration. 2.2.2 IAA production Indole-3-acetic acid (IAA) synthesis by bacteria was determined using the Salkowski method (Do et al., 2023). Bacteria were cultured in 25 ml of LB medi- um supplemented with 0.1 g l-1 tryptophan, incubated at 30  °C for 48 hours with shaking at 120 rpm. The IAA content in the supernatant was quantified using the Salkowski reagent with colorimetric measurement at 530 nm and IAA as the standard. 2.2.3 Phosphate solubilization Phosphorus solubilization capability was assessed by culturing bacteria in 100 ml National Botanical Research Institute’s phosphate growth (NBRIP) liquid medium at room temperature for 7 days with agitation at 120 rpm (Yanlei and Xiaoping, 2018). The super- natant was collected by centrifugation at 10,000 rpm for 15 minutes at 4  °C. Solubilized phosphorus was quantified using the molybdenum blue method with ammonium molybdate at 880 nm and KH2PO4 as the standard. 2.2.4 Molecular identification of bacterial isolate The molecular method was used to identify the species of strain TL8. Bacteria were grown overnight in LB broth at 30 °C with shaking (120 rpm). Genomic DNA was extracted using the NucleoSpin® Tissue extraction kit (Macherey-Nagel, Germany). The 16S rRNA gene was amplified via PCR with the primer pair 27F and 1492R. PCR conditions were: initial denaturation at 94  °C for 2 minutes, followed by 35 cycles of 94  °C for 30 seconds, 55  °C for 20 seconds, 72  °C for 1 minute, final extension at 72  °C for 5 minutes, and hold at 4 °C. PCR products were cleaned and sequenced by 1st BASE Company (Malaysia). The 16S rRNA gene sequence of strain TL8 was compared to sequences in the GenBank database. 2.3 EVALUATION OF ENDOPHYTIC BACTERIA ON CASSAVA PLANT GROWTH UNDER GREENHOUSE CONDITIONS The greenhouse experiment was conducted in the greenhouse of the Vietnam National University of For- estry (VNUF) during the winter-spring cropping sea- son of 2022-2023 (November 2022 - May 2023) using cassava cuttings (15-20 cm). Soil from Tuy Lai commune, My Duc district, Ha Noi, Vietnam, was used. The soil analysis crite- ria include pH and EC, extracted with distilled water at a ratio of 1:2.5 (soil: water). pH is measured with a pH meter, and EC is measured with an EC meter. Available phosphorus (using the Bray II method) is determined by extracting soil with 0.1N HCl + 0.03N NH4F at a soil-to-water ratio of 1:7, then measured on a spectrophotometer at a wavelength of 880 nm. Ex- changeable potassium is extracted using 0.1M BaCl2 and measured with an atomic absorption spectrom- eter. Soil texture composition is determined using the Robinson pipette method. The properties of soil were pHKCl 4.69, EC = 1.91 mS cm -1, total N 0.11%, organic matter 3.02 %, soluble P2O5 = 20.12 mg P kg -1, soluble K2O = 0.27 meq 100g -1. The bacterial strains with strong abilities to fix nitrogen, solubilize phosphate, and produce IAA were chosen. The selected bacteria strains were cul- tured in LB media at 30 °C for 24 hrs. The culture was centrifuged at 8000 rpm for 10 minutes at 4 °C, and the cells were resuspended in sterilized water to OD600 = 1.0. Cassava cuttings (15-20 cm) were disinfected and immersed in bacterial suspension for 1 hour before planting in pots (0.3 m × 0.4 m) filled with prepared soil (Zhang et al., 2022). The experiment followed a two-factor completely Table 1: Summary of treatment combination under green- house conditions Urea fertilizer Without bacterial inoculation With bacterial inoculation of strain TL4 With bacterial inoculation of strain TL8 No urea fertilizer T1 T5 T9 30 kg urea ha-1 T2 T6 T10 60 kg urea ha-1 T3 T7 T11 90 kg urea ha-1 T4 T8 T12 Acta agriculturae Slovenica, 120/4 – 20244 Q. T. DO et al. randomized block design: factor A (urea fertilizer lev- els: 0, 30, 60, and 90 kg ha-1) and factor B (bacterial strains: with and without bacteria) with three replica- tions (Table 1). Fertilizers used were urea (46 % N), Lam Thao su- perphosphate (16 % P2O5), and Kali Phu My MOP (60 % Growth parameters (plant height, number of leaves, di- ameter of base, trunk, and stem) were measured at 90 days after planting (DAP), and tuber yield, number of tubers, tuber length, and tuber diameter were meas- ured at harvest. The Kjeldahl method was applied to determine the total nitrogen content in cassava plants’ leaves, roots, and stems from T1 (without nitrogen fertilizer and bacterial TL8 inoculation) and T9 (with bacterial TL8 inoculation only) experiments. Approximately 0.5 g of dried and finely ground plant tissue from each part was placed in a digestion flask. To each sample, 10 ml of concentrated H₂SO₄ was added along with 0.5 g of a catalyst mixture consisting of K₂SO₄ and se- lenium (0.005 g). The digestion process was done by heating the mixture until the solution became clear, indicating the complete breakdown of organic matter. After digestion, the solution was cooled and diluted with 50 ml of distilled water. To neutralize the acid and convert ammonium ions (NH₄+) to ammonia (NH₃), 40 ml of 10 mol l-¹ NaOH was added to the mixture. The ammonia was then distilled into a receiving flask containing 25 ml of 2 % boric acid solution, with the distillation process continuing until approximately 100 ml of distillate had been collected. The collected distillate was titrated with 0.1 mol l-¹ HCl until the endpoint was reached, as indicated by a color change of the pH indicator. The volume of HCl used in the titration was recorded, and the total nitrogen content was calculated based on the titration results. This pro- cedure allowed for the accurate quantification of total nitrogen in the different plant tissues according to the Kjeldahl method described by Bremner (1996). 2.4 EVALUATION OF PROMISING ENDOPHYTIC BACTERIA ON CASSAVA PLANT GROWTH UNDER FIELD CONDITIONS The field experiment was conducted during the summer-autumn crop of 2023 at Tuy Lai commune, My Duc district, Ha Noi, Vietnam. The soil was pre- pared by plowing to a depth of 15-20 cm and arrang- ing into beds (100 cm width, 50 cm height, 10 m length) with 30 cm spacing between beds. Cuttings were planted in single rows per bed, with 80 cm spac- ing between cuttings. The experiment followed a completely rand- omized block design with one factor, comprising 8 different treatments, each replicated three times (sum- marized in Table 3). Fertilizer application and growth monitoring were conducted as in the greenhouse ex- periment (see Table 2). 2.5 DATA ANALYSIS Data were statistically analyzed using Excel and IRRISTAT software. Tukey’s Honestly Significant Dif- ference (HSD) tests were used for pairwise compari- sons, maintaining a 95 % confidence level. Fertilization period Describe On the day of planting Apply whole phosphate fertilizer 1st application (25 DAP) Apply 1/3 urea fertilizer + 1/3 potassium fertilizer 2nd application (50 DAP) Apply 1/3 urea fertilizer + 1/3 potassium fertilizer 3rd application (80 DAP) Apply all the remaining urea and potassium fertilizer Table 2: Period and dosage of fertilizer for experiments DAP: day after planting Treatments Describe F1 Without Urea fertilizer and bacterial inoculation F2 With 30 kg urea ha-1 F3 With 60 kg urea ha-1 F4 With 90 kg urea ha-1 F5 Without urea fertilizer + with bacterial inocu- lation of TL8 F6 With 30 kg urea ha-1+ with bacterial inocula- tion of TL8 F7 With 60 kg urea ha-1+ with bacterial inocula- tion of TL8 F8 With 90 kg urea ha-1+ with bacterial inocula- tion of TL8 Table 3: Summary of treatment combination under field conditions Acta agriculturae Slovenica, 120/4 – 2024 5 Utilization of nitrogen-fixing endophytic bacteria to ... yield and nitrogenous nutrient uptake of Cassava plant (Manihot esculenta Crantz) 3 RESULTS AND DISCUSSION 3.1 ISOLATION AND CHARACTERIZATION OF ENDOPHYTIC BACTERIA FROM CASSAVA Eleven strains of endophytic bacteria (TL1 to TL11) were isolated from root, stem, and leaf samples of native cassava (Table 4). Two strains were obtained from stem samples, four from leaf samples, and six from root samples. The isolated bacterial strains pre- dominantly displayed round colonies with small sizes ranging from 0.2 to 0.6 cm, appearing glossy, convex, and translucent white. Research into endophytic bacteria from cassava plants has demonstrated their significant potential in promoting plant growth and health through various mechanisms, such as nitrogen fixation, phosphate sol- ubilization, and IAA production (Ferreira et al., 2021; Feng et al., 2023; Ferreira et al., 2024). In terms of nitrogen fixation, only four isolat- ed bacterial strains produced NH4 +, with strain TL8 showing the highest production at 14.95 mg l-1. This NH4 + production was higher than that reported by Zhang et al., (2022), where strain A02 produced 13.38 mg l-1. Therefore, strain TL8 might play a critical role in providing nitrogenous compounds for plant devel- opment. Phosphate solubilization is another critical trait observed in cassava-associated endophytic bacteria. Many isolates could solubilize inorganic phosphate, enhancing phosphorus availability in the soil, which is essential for plant growth. This trait is particularly beneficial for cassava, often cultivated in low-fertility soils with limited phosphorus availability (Omondi et al., 2018; Zhang et al., 2022; Feng et al., 2023; Ferreira et al., 2024). Strains TL4 (15.51 mg l-1) and TL8 (14.27 mg l-1) showed the highest phosphorus solubilization. According to Zhang et al. (2022), strain A02 produced 101.23 mg l-1 of phosphorus solubilization in 8 days after incubation. The data suggest that strains TL4 and TL8 present significant promise for the biofertilizer industry. Additionally, the ability of endophytic bacteria to produce IAA, a plant hormone regulating growth and development, has been well-documented (Zhang et al., 2022; Feng et al., 2023; Ferreira et al., 2024). Many isolates, especially from cassava roots, showed positive results for IAA production (Ferreira et al., 2021; Zhang et al., 2022). IAA stimulates root elonga- tion and improves nutrient uptake, supporting overall plant growth and resilience. Six endophytic bacterial strains in our study synthesized IAA, with the highest synthesis by TL8 (46.57 mg l-1), followed by TL7 (30.51 mg l-1) and TL3 (21.15 mg l-1). The lowest IAA syn- thesis was by TL1 at 11.23 mg l-1. Ferreira et al. (2021) reported strain A02 produced 1.56 mg l-1 of IAA in 2 days. These results indicate that strain TL8 could po- tentially be applied to produce IAA, promoting plant growth. Based on their superior abilities in nitrogen fixa- tion, phosphorus solubilization, and IAA synthesis, strains TL4 and TL8 were selected for further study. Isolates Source Amount of NH4 + (mg l-1) Amount of soluble PO4 3- (mg l-1) Amount of IAA (mg l-1) TL1 Stem - - 11.23 ± 0.08 d TL2 - 6.43 ± 0.12 cd - TL3 Leaf - - 21.15±0.04 c TL4 4.31 ± 0.11 b 15.51 ± 0.09 a 15.12 ± 0.05 d TL5 - - - TL6 3.24 ± 0.03 c 8.71 ± 0.12 c - TL7 Root - 3.09 ± 0.13 e 30.51 ± 0.09 b TL8 14.95 ± 0.21 a 14.27 ± 0.21 b 46.57 ± 0.11 a TL9 - - - TL10 3.21 ± 0.04 c 3.12 ± 0.11 e 14.23 ± 0.07 d TL11 - 5.25 ± 0.21 d - Table 4: Characteristics of endophytic bacteria isolated from the cassava plant Data present means ± SD (n = 3). Values in the same column with the same letter(s) are not significantly different as determined by the least signifi- cant difference (HSD) test (p < 0.05). Acta agriculturae Slovenica, 120/4 – 20246 Q. T. DO et al. Molecular identification indicated that TL4 and TL8 were closely related to Burkholderia cenocepacia and Priestia aryabhattai, with percentage identities of 98.85  % and 98.74  %, respectively. The sequences were deposited in GenBank with accession numbers PQ113673 and PQ119839. These endophytic bacterial strains were further studied under greenhouse and field conditions to highlight their potential as biofertilizers in sustainable agriculture practices. 3.2 EFFECTS OF ENDOPHYTIC BACTERIAL STRAIN TL4 AND TL8 COMBINED WITH NITROGEN FERTILIZER DOSES ON THE GROWTH AND YIELD OF CASSAVA UNDER GREENHOUSE CONDITIONS The results of this study underscore the significant role that nitrogen fertilization and bacterial inoculation play in the growth and yield of cassava under greenhouse conditions. At 90 days after planting (DAP), statistical- ly significant differences in plant height were observed between the nitrogen fertilization and bacterial inocu- lation experiments (Figure 1A). Plant height in the ni- trogen fertilization experiments ranged from 110.13 to 153.19 cm, with the lowest heights in the non-nitrogen- fertilized plants. Among the bacterial inoculation treat- ments, plant height ranged from 122.34 to 151.26 cm, with the highest in plants inoculated with strain TL8 (151.26 cm). Additionally, the number of leaves (Figure 1B), base diameter (Figure 1C), trunk diameter (Figure 1D), and stem diameter (Figure 1E) showed statistically significant differences among the nitrogen fertilization treatments. The lowest number of leaves, base diameter, trunk diameter, and stem diameter were observed in the non-nitrogen fertilized plants. This is because nitrogen significantly influences cassava growth and development, promoting robust leaf, stem, and root development com- pared to nitrogen-deficient plants (Rafikova et al., 2016; Xing et al., 2016). Moreover, the concentrations of total N in cassava plants’ leaves, stems, and roots inoculated with endo- phytic bacteria TL8 were increased compared to the control. Total N in the stems and roots did not statisti- cally differ between experiments; however, total N in the leaves differed, and the inoculation of TL8 had the highest concentrations (Table 5). These results are in agreement with the results of previous reports (Szilagyi- Zecchin et al., 2014; Li et al., 2017) which demonstrated that cassava stems inoculated with endophytic bacteria led to a higher amount of nitrogen content in the leaves. Nitrogen uptake is crucial for cassava productivity as it enhances photosynthesis, supports protein and enzyme synthesis, and maintains a balanced carbon-to-nitrogen ratio. Adequate nitrogen improves chlorophyll content, promotes root development, and leads to greater tuber biomass and starch accumulation (Feng et al., 2023). Studies confirm that nitrogen-deficient cassava shows reduced growth and yield, while optimal nitrogen fertili- Figure 1: Effects of endophytic bacterial strains TL4 and TL8 combined with the doses of nitrogen fertilizer on (A) cassava plant height; (B) number of leaves; (C-E) Diameter of base, trunk, and stem at 90 DAP under greenhouse conditions. Plot- ted data present means ± SD (n = 3), the different letter(s) in- dicate significant differences as determined by the least signifi- cant difference (HSD) test (p < 0.05). Acta agriculturae Slovenica, 120/4 – 2024 7 Utilization of nitrogen-fixing endophytic bacteria to ... yield and nitrogenous nutrient uptake of Cassava plant (Manihot esculenta Crantz) zation improves nutrient use efficiency, boosting produc- tivity sustainably (Feng et al., 2023). Bacterial inoculation with strain TL8 led to the highest plant height (151.26 cm), suggesting superior nitrogen-fixation capabilities compared to strain TL4. This finding aligns with studies highlighting the potential of specific endophytic bacterial strains to enhance plant growth by improving nitrogen availability (Zhang et al., 2022). The observed statistical differences in the number of leaves, base diameter, and stem diameter further sup- port the importance of adequate nitrogen supply (Feng et al., 2023). Tuber characteristics, including the number of tu- bers per pot (Figure 2A), tuber diameter (Figure 2B), and tuber length (Figure 2C), differed statistically among the nitrogen fertilization treatments. The combination of 60 kg urea ha-1 with bacterial inoculation showed significant differences compared to the no-nitrogen treatment and the 30 kg urea ha-1 treatment with bacterial inoculation, but not from the 90 kg urea ha-1 treatment combined with bacterial inoculation. Among the bacterial inocula- Treatment Nitrogen content (mg g-1) Leaves Stems Roots With bacterial TL8 inoculation only 46.87 ± 0.43a 13.58 ± 0.51a 13.08 ± 0.61a Without nitrogen fertilizer and bacterial TL8 inoculation 35.96 ± 0.74b 13.04 ± 0.46a 12.73 ± 0.58a Table 5: Effect of endophytic bacteria TL8 on the nitrogen content of cassava plant in a greenhouse experiment Data present means ±SD (n = 3). Values in the same column with the same letter(s) are not significantly different as determined by the least signifi- cant difference (HSD) test (p < 0.05). Figure 2: Effects of endophytic bacterial strains TL4 and TL8 combined with the doses of nitrogen fertilizer on (A) the number of tubers per 10 m2; (B) Length of tubers; (C) Diameter of tuber; and (D) Yield at harvest under greenhouse conditions. Plotted data present means ± SD (n = 3), the different letter(s) indicate significant differences as determined by the least significant difference (HSD) test (p < 0.05). Acta agriculturae Slovenica, 120/4 – 20248 Q. T. DO et al. tion treatments, significant differences were observed for the number of tubers per pot, tuber diameter, and tuber length, with the highest values for these parameters in the TL8 inoculation. Tuber yield per pot also differed significantly be- tween the nitrogen fertilization and bacterial inoculation treatments (Figure 2D), with yields ranging from 254.37 to 640.94 g pot-1. The lowest yield was observed without nitrogen fertilization (254.37 g pot-1), and the lowest yield among bacterial inoculation treatments was with- out bacterial inoculation (441.69 g pot-1). Providing suffi- cient nitrogen enhances cassava growth and increases tu- ber yield (Uwah et al., 2013; Zhang et al., 2022). Among the two experimental bacterial strains, TL8 increased the number of tubers, tuber diameter, and tuber length, enhancing cassava yield compared to TL4. This superior performance may be attributed to TL8’s higher nitrogen- fixation activity, IAA production, and phosphate solubili- zation, which enhance nutrient availability and uptake by the plant (Biswas et al., 2022; Argotte-Ibarra et al., 2022; Do et al., 2023). Overall, this study demonstrates that nitrogen ferti- lization and bacterial inoculation, particularly with strain TL8, significantly improve cassava growth and yield un- der greenhouse conditions. Further research exploring the long-term effects of these treatments with strain TL8 under field conditions is necessary to refine cassava cul- tivation practices. 3.3 EFFICACY OF BACTERIA TL8 ON THE GROWTH AND YIELD OF CASSAVA UNDER FIELD CONDITIONS At 90 DAP, plant heights across the experiments were statistically different (Figure 3A), ranging from 108 to 153 cm. The application of 60 kg urea ha-1 combined with TL8 inoculation (F6) produced plant heights similar to those achieved with 90 kg urea ha-1 without bacterial inoculation (F7). The lowest plant height was observed in treatments without nitrogen fertilization and bacte- rial inoculation (F1). The number of leaves also showed statistically significant differences (Figure 3B), with the lowest number (30.28 leaves) in the non-nitrogen ferti- lization experiments (F1). These data indicate the poten- tial application of strain TL8 to enhance the nitrogen ef- ficiency of cassava plants under field conditions. Base and trunk diameters were significantly dif- ferent (Figures 3C and 3D), with the highest diameters found in the treatments with 60 kg urea ha-1 + strain TL8 (F6), 90 kg urea ha-1 + no bacterial inoculation (F7), and 90 kg urea ha-1 + strain TL8 (F8). This aligns with previ- ous research demonstrating that adequate nitrogen sup- ply, especially when combined with beneficial bacterial inoculants, can significantly enhance structural growth parameters (Biswas et al., 2022; Zhang et al., 2022, Aasfar et al., 2024). The diameter of the stem across the experiments did not show any statistically significant differences, rang- ing from 0.79 to 0.88 cm (Figure 3E). This consistency suggests that while nitrogen and bacterial treatments influence overall plant height and biomass, they might not significantly alter certain morphological traits under the given experimental conditions. Similar findings have been reported in studies on other crops, where the im- pact of nitrogen-fixing bacteria was more pronounced on overall growth and yield metrics rather than specific morphological characteristics (Aasfar et al., 2024). Ad- ditionally, applying 60 kg urea ha-1 combined with TL8 bacterial inoculation (F6) resulted in cassava growth not statistically different from that achieved with 90 kg urea ha-1 without bacterial inoculation (F7). This is particu- larly relevant for sustainable agriculture, where reducing the dependency on chemical fertilizers can have signifi- cant environmental benefits (Do et al., 2023; Aasfar et al., 2024). The ability of TL8 to enhance nitrogen efficiency could lead to more sustainable cassava production prac- tices, aligning with global efforts to minimize agricultur- al inputs while maintaining high yields. Further supporting these findings, previous research has shown that the application of nitrogen-fixing bacte- ria can lead to increased plant height and dry weight in various crops, highlighting their role in improving nitro- gen availability and utilization (Xu et al., 2018; Do et al., 2023; Aasfar et al., 2024). These studies suggest that the beneficial effects of bacterial inoculants are not limited to cassava but extend to other rooted crops as well. The number of tubers differed significantly among the experiments (Figure 4A), ranging from 31.35 to 58.17 tubers. The lowest number of tubers (31.35) was observed in the no-nitrogen fertilization treatment (F1). This find- ing aligns with the general understanding that nitrogen is a key nutrient for promoting tuber growth and overall plant productivity (Zhang et al., 2022; Aasfar et al., 2024). Tuber length also showed statistically significant differences, with the highest lengths recorded in the treatments including F6 (60 kg urea ha-1 + TL8 inocula- tion), F7 (90 kg urea ha-1 without bacterial inoculation), and F8 (90 kg urea ha-1 + TL8 inoculation) (Figure 4B). Similarly, tuber diameter varied significantly at the 5 % level, ranging from 4.55 to 5.48 cm, with the smallest di- ameter (4.55 cm) in the F1 treatment (Figure 4C). These results highlight the beneficial effects of adequate nitro- gen supply, whether through fertilization or bacterial in- oculation, on tuber size and quality. Moreover, the results indicated that applying 60 kg urea ha-1 combined with Acta agriculturae Slovenica, 120/4 – 2024 9 Utilization of nitrogen-fixing endophytic bacteria to ... yield and nitrogenous nutrient uptake of Cassava plant (Manihot esculenta Crantz) strain TL8 (F6) achieved similar numbers, lengths, and diameters of tubers as the 90 kg urea ha-1 without bacteri- al inoculation (F7). This suggests that the nitrogen-fixing ability of strain TL8 contributed additional nitrogen to the cassava plants, allowing the F6 treatment to match the performance of the F7 treatment. Previous research supports these findings, showing that nitrogen-fixing microbial fertilizers can enhance plant growth and yield (Zhang et al., 2022; Aasfar et al., 2024). Combining nitrogen fertilization with bacterial inoculation significantly impacts cassava tuber yield, demonstrating the potential for optimizing fertilizer use while maintaining high productivity. Figure 4D indicates that tuber yield did not differ significantly between the treatments of F7 (18.49 t ha-1), F6 (18.42 t ha-1), and F8 (19.15 t ha-1). However, these treatments showed a 1.12% higher yield compared to the no-nitrogen treatments (F1 at 9.83 t ha-1 and F2 at 10.86 t ha-1), as well as the F4 (15.93 t ha-1), F3 (13.69 t ha-1), and F5 (15.79 t ha-1) treatments. These results suggest that using 60 kg urea ha-1 combined with strain TL8 achieved similar yields to the application of 90 kg urea ha-1 without bacterial inoculation, indicat- Figure 3: Effects of bacterial strain TL8 combined with the doses of nitrogen fertilizer on (A) cassava plant height; (B) number of leaves; (C-E) Diameter of base, trunk, and stem at 90 DAP under field conditions. F1: without urea fertilizer and bacterial inocula- tion; F2: without urea fertilizer and with bacterial strain TL8; F3: with 30 kg urea ha-1 and without bacterial strain TL8; F4: with 30 kg urea ha-1and with bacterial strain TL8; F5: with 60 kg urea ha-1 and without bacterial strain TL8; F6: with 60 kg urea ha-1 and with bacterial strain TL8; F7: with 90 kg urea ha-1 and without bacterial strain TL8; F8: with 90 kg urea ha-1 and with bacterial strain TL8. Plotted data present means ± SD (n = 3), the different letter(s) indicate significant differences as determined by the least significant difference (HSD) test (p < 0.05). Acta agriculturae Slovenica, 120/4 – 202410 Q. T. DO et al. Figure 4: Effects of bacterial strain TL8 combined with nitrogen fertilizer doses on (A) the number of tubers per 10 m2; (B) Length of tubers; (C) Diameter of tuber; and (D) Yield at harvest under field conditions. F1: without urea fertilizer and bacterial inocula- tion; F2: without urea fertilizer and with bacterial strain TL8; F3: with 30 kg urea ha-1 and without bacterial strain TL8; F4: with 30 kg urea ha-1and with bacterial strain TL8; F5: with 60 kg urea ha-1 and without bacterial strain TL8; F6: with 60 kg urea ha-1 and with bacterial strain TL8; F7: with 90 kg urea ha-1 and without bacterial strain TL8; F8: with 90 kg urea ha-1 and with bacterial strain TL8. Plotted data present means ± SD (n = 3), the different letter(s) indicate significant differences as determined by the least significant difference (HSD) test (p < 0.05). ing that a reduction of 30 kg urea ha-1 could be feasible for cassava cultivation. This finding aligns with studies by Zhang et al. (2022) and Aasfar et al. (2024), which sug- gest that nitrogen-fixing bacteria can reduce the reliance on nitrogen fertilizers, emphasizing their secondary role when effective bacterial inoculation is applied. The observed results underscore the benefits of inte- grating nitrogen-fixing bacterial inoculants into fertiliza- tion practices. By reducing the reliance on chemical fer- tilizers, farmers can achieve cost savings and minimize environmental impacts, such as nitrogen leaching and soil degradation. This approach aligns with sustainable agricultural practices and supports the shift toward more eco-friendly farming methods (Zhang et al., 2022). 4 CONCLUSIONS The integration of nitrogen-fixing endophytic bac- teria, specifically strain TL8, with nitrogen fertilization demonstrates significant potential for enhancing cassava growth and yield. The study’s findings highlight the supe- rior performance of TL8 in nitrogen fixation, phosphate solubilization, and IAA synthesis, contributing to im- proved plant height, leaf number, and tuber yield under both greenhouse and field conditions. The ability of TL8 to achieve similar yields with reduced nitrogen fertiliza- tion (60 kg urea ha-1) as conventional higher fertilization rates (90 kg urea ha-1) underscores its role in promoting sustainable agricultural practices. This approach not only enhances crop productivity but also reduces the reliance on chemical fertilizers, offering environmental and eco- nomic benefits. Further research is warranted to explore the long-term impacts and optimize the application of these bacterial inoculants in diverse agricultural settings. 5 REFERENCES Argotte-Ibarra, L., Barreiro-Quino, O.F., Carlos, A.R., José, A.H.M., Hans, T.C.S. (2022). Analysis of the solubil- ity of phosphate rock from Aipe (Colombia) via forma- tion of 2Na-EDTA complex. 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