Unraveling the Novel Synergistic Effects of Crop Rotation and Rhodopseudomonas palustris Inoculation on Rice Productivity and Soil Nutrient Dynamics

Unraveling the Novel Synergistic Effects of Crop Rotation and Rhodopseudomonas palustris Inoculation on Rice Productivity and Soil Nutrient Dynamics | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Unraveling the Novel Synergistic Effects of Crop Rotation and Rhodopseudomonas palustris Inoculation on Rice Productivity and Soil Nutrient Dynamics Laurence Shiva Sundar, Kuei-Shan Yen, Yao-Tsung Chang, Yun-Yang Chao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3383462/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and Aim The use of beneficial bacteria, such as purple non-sulfur bacteria (PNSB), has shown great potential for improving plant growth and agricultural production. However, the full extent of their benefits and interaction with agricultural practices is yet to be fully understood. The present study aimed to investigate the synergistic effects of PNSB and crop rotation on rice growth and yield in a field setting and to explore the underlying plant and soil mechanisms by which these practices can benefit farming systems. Methods The experiment was conducted over two rice cropping seasons, with djulis grown between the rice crops as a rotation crop. Data on the growth and yield of rice was collected and statistically analyzed using a one-way analysis of variance (ANOVA) and Duncan's multiple range test. Results The study demonstrates that PNSB treatment significantly increased the concentration of 5-aminolevulinic acid (5-ALA) in plants, indicating enhanced photosynthesis. Moreover, when combined with crop rotation, PNSB remarkably improved soil fertility. These combined benefits resulted in substantial increases in tiller numbers (163%), leaf chlorophyll content (13%), and lodging resistance (66%) compared to the control. The combined treatment also resulted in higher productive tillers per hill (98%), average grain per hill (106%), and grain fertility (37%). This led to increased grain yield (104%), shoot dry weight (28%), and harvest index (54%). Conclusion Our study indicates that a combined approach of PNSB inoculation and crop rotation can effectively enhance the growth and yield of rice plants. These findings have significant implications for sustainable rice production and could potentially contribute to addressing global food security challenges. Improving plant growth and yield could help meet the increasing demand for rice in the face of a growing global population. 5-aminolevulinic acid agricultural production agricultural practices antioxidant enzymes food security photosynthetic bacteria plant and soil mechanisms sustainable rice production Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Rice ( Oryza sativa L.) crop is a vital staple that plays a crucial role in providing food and income for millions of people worldwide (Nguyen and Ferrero 2006 ; Khush 2013 ; Prasad et al. 2017 ; Kumar et al. 2022 ). However, its sustainability and productivity face increasing threats from various challenges (Kumar et al. 2022 ). For instance, the excessive application of chemical fertilizers and pesticides to meet the high demand for rice has resulted in numerous problems, including escalated production costs, environmental deterioration, and adverse impacts on human health (Prashar and Shah 2016 ; Sharma and Singhvi 2017 ; Baweja et al. 2020 ; Srivastav 2020 ). Similarly, the conventional monoculture practice of rice cultivation has also given rise to challenges such as soil quality and fertility depletion, disease outbreaks, pest infestations, and declining yields (Berg 2002 ; He et al. 2016 ; Goulart et al. 2020 ). Therefore, these issues underscore the necessity for alternative and more sustainable farming practices, such as crop rotation, rice-fish cultivation, integrated pest management methods, and organic farming practices. Crop rotation, an ancient agricultural practice, has been used for centuries to sustainably improve soil quality and crop yield. It refers to the systematic approach of cultivating different crops on the same agricultural land in a planned sequence. Studies have shown that rotating crops helps to enhance soil fertility, regulate pests and diseases, and increase yield (Karlen et al. 1994 ; Dadhich et al. 2015 ; Kakraliya et al. 2018 ; Kumar et al. 2020b ). The effectiveness and advantages of crop rotation have been widely acknowledged. For example, in rice-based cropping systems, crop rotation helps to improve soil quality, reduce pests and diseases, and enhance yield (Huang et al. 2020 ; Kumar et al. 2020a ; Adelana et al. 2022 ). Additionally, the photosynthetic bacterium Rhodopseudomonas palustris species has been observed to fix nitrogen while producing compounds aiding plant growth, such as 5-aminolevulinic acid (5-ALA) (Kantha et al. 2015 ; Kantachote et al. 2016 ). It has been demonstrated that the use of R. palustris can increase the growth and yield of a variety of crops, including pak choi (Wong et al. 2014 ; ShuHua et al. 2015; Xu et al. 2016 ), stevia (Xu et al. 2018 ), tobacco (JianFeng et al. 2014 ; Su et al. 2017 ), mushroom (Han 1999 ), Chinese dwarf cherry (Yin et al. 2012 ), bean (Batool and Rehman 2017 ), and rice (Harada et al. 2005 ; Kantha et al. 2015 ; Kantachote et al. 2016 ; Nookongbut et al. 2018 ; Yen et al. 2022 ; Khuong et al. 2022 ; Iwai et al. 2022 ). In addition, R. palustris can act as a biofertilizer, reducing the need for chemical fertilizers while boosting soil health, crop yield, and nutrient assimilation efficiency (Kantha et al. 2015 ; Kantachote et al. 2016 ; Wang et al. 2021b ). Numerous studies have looked at the individual effects of crop rotation (Tanveer et al. 2019 ; Costa et al. 2020 ; Yu et al. 2022 )d palustris inoculation (Wong et al. 2014 ; Hsu et al. 2021 ; Yen et al. 2022 ) on rice growth and yield; however, their combined effects have not been thoroughly examined. The combined effects of R. palustris inoculation and crop rotation on rice yield and growth may have significant implications for developing environmentally friendly and economically viable crop management techniques. Therefore, the current study aims to assess the combined impact of crop rotation and R. palustris inoculation on the growth and yield of rice crops in field conditions. Additionally, the study will investigate how R. palustris inoculation and crop rotation affect antioxidant enzyme activity and 5-ALA levels, which are crucial markers of plant growth and stress. In particular, this research will deepen our understanding of how crop rotation and R. palustris inoculation interact to affect rice productivity and growth and offer suggestions for establishing environmentally sound and long-lasting rice farming practices. Materials and methods Experimental design and setup The current research was conducted at the Practice Farm of the Department of Plant Industry, National Pingtung University of Science and Technology (NPUST), Taiwan, R.O.C. The farm is situated in an open area at coordinates 22°38'54.0" N and 120°37'01.9" E. Two fields, each measuring approximately 21 m in length and 9 m in width, were selected for the study area. The blocks were established parallel to each other in the same area under comparable environmental conditions. During the first year of the study, which took place between January and May 2022 (the primary rice-growing season in Taiwan), two fields were utilized to cultivate rice crop. One field was designated as the control group, while the other served as the treatment group. Following rice cultivation, djulis was cultivated in two separate fields from September to December (the primary season for djulis cultivation) of the same year. After djulis were harvested, the stems were crushed into smaller pieces and spread in the soil before final land preparation, as shown in Fig. 1 . Additionally, in order to enhance soil fertility, any remaining plant materials, including roots, were thoroughly incorporated into the soil through rotovating, ensuring a more balanced nutrient composition and promoting favorable soil conditions for optimal rice growth. In the second year of the study, rice was once again cultivated from January to May 2023. All management practices, including land preparation, planting, and harvesting, were performed uniformly in each block to prevent biases in the results. The Kaohsiung 147 rice crop was transplanted at the 5-leaf stage using the rice transplanter in each field. Ten plants were randomly tagged for field data collection four weeks after transplanting (WAT), while 4 random plants were selected to analyze antioxidant enzyme activities and 5-ALA concentration. The weather conditions, such as air temperature, relative humidity, and light intensity, were monitored using the fully automated KLIMALOG Microclimate Environment Monitoring System provided by Taiwan Hibot Co., Ltd., Kaohsiung, Taiwan R.O.C. Additionally, during the second trial, we extensively monitored the soil environment, specifically focusing on soil temperature and soil electrical conductivity (EC). These parameters were carefully measured using AgriWeather Field Sensor (Beehive Data Technology Co., Ltd., Taipei, Taiwan R.O.C.) to gain insights into the below-ground conditions and their potential impact on rice growth. The data were obtained weekly from the online system and recorded to make informed decisions on management practices. In addition, the soil nutrient analysis was conducted to understand the changes in the soil nutrients under different treatments. Preparation and application of PNSB The biofertilizer containing the R. palustris species of PNSB was prepared using the initial stock obtained from the Food Industry Research and Development Institute (FIRDI), Taiwan R.O.C (research number PSB32). The culture medium was formulated based on the method described by Lee et al. ( 2016 ) with some modifications as per the available materials and the suggestions provided by FIRDI. The bacteria were cultivated in a 20 L transparent water bottle and placed in the greenhouse under indirect sunlight for two weeks to promote optimal growth, indicated by the development of a dark maroon color, as shown in Fig. 2 . The culture bottle was inspected and agitated daily to ensure uniform dispersion of the culture medium for consistent bacterial growth. After 14 days of culture, 10 mL of the stock solution was sampled for laboratory analysis to determine the colony-forming unit (CFU). The original CFU count was determined by the standard plate count (SPC) technique and was adjusted to 2.46 x 10 8 to suit the experimental requirements. The CFU adjustment was implemented to ensure sufficient PNSB in the inoculum, capable of significantly impacting plant growth and yield. This decision was based on previous studies demonstrating the efficacy of similar concentrations of the same bacterial species in promoting plant growth and yield improvement (Yoshida et al. 1991 ; Han 1999 ; Harada et al. 2005 ; Koh and Song 2007 ; Lee et al. 2008 ; Yin et al. 2012 ; Fan et al. 2012 ; Wu et al. 2013 ; Wong et al. 2014 ; Hua et al. 2014 ; ShuHua et al. 2015; Su et al. 2017 ; Nookongbut et al. 2018 ; Xu et al. 2018 ). At 4 WAT, rice plants were inoculated with PNSB, and the inoculation was repeated every two weeks until the early reproductive (heading) stage. Antioxidant enzyme activity analysis The antioxidant enzyme activity was analyzed from WAT 4 to WAT 12 weekly. The enzymes analyzed were ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), and superoxide dismutase (SOD). The protein content of the enzyme extract was determined using the method of Bradford ( 1976 ). Samples collected in the field were immediately placed in dry ice and transported to the laboratory for analysis. In the laboratory, a fresh leaf sample (0.05 g) was ground using liquid nitrogen and then homogenized with sodium phosphate buffer (50 mM; pH 6.8 for APX, CAT, GR, and 50 mM; pH 7.4 for SOD) for further grinding before being placed in an ice bath. The solution was then centrifuged at 12,000x g for 20 min (APX, CAT, GR) and 15,000x g for 30 min (SOD) using a Velocity 14R refrigerated Centrifuge (Dymamica Scientific Ltd., Livingston UK) at 4 ℃, and the supernatant was collected. The APX activity was analyzed using the method of Nakano and Asada ( 1981 ). The absorbance was measured at 290 nm for 1 min using a Double Beam U-2900 Spectrophotometer (Hitachi High-Tech Corporation, Japan). As the concentration of ascorbate (AsA) decreased, the absorbance at 290 nm also reduced, and the extinction coefficient of AsA (2.8 mM − 1 cm − 1 ) was used to calculate the APX activity. One unit of APX was defined as the amount of enzyme needed to degrade 1 mole of AsA in 1 min. The CAT activity was analyzed using the method of Kato and Shimizu ( 1987 ). The reduction in hydrogen peroxide amount was measured at 240 nm, and the extinction coefficient (40 mM − 1 cm − 1 ) was used to calculate CAT activity. One unit of CAT was defined as the amount of enzyme needed to degrade 1 mole of hydrogen peroxide in 1 min. The GR activity was analyzed using the method of Foster and Hess ( 1980 ). One unit of GR was defined as the amount of enzyme needed to decrease the absorbance at 340 nm at 1 min. Finally, the SOD activity was analyzed using the method by Paoletti et al. ( 1986 ). One unit of SOD was defined as the amount of enzyme that inhibited the rate of NADH oxidation by 50% in the blank sample. Analysis of 5-aminolevulinic acid The method by Mauzerall and Granick ( 1956 ), with slight modifications, was used to determine the concentration of 5-ALA. Initially, a leaf sample weighing 0.05 g was homogenized with sodium acetate buffer (1M; pH 4.7) using a mortar and pestle in an ice bath. The solution was then centrifuged at 10000x g for 5 min at 4°C using a Velocity 14R Refrigerated Centrifuge (Dymamica Scientific Ltd., Livingston, UK), and the resulting supernatant was collected. To this, a 1 mL aliquot of the supernatant was mixed with 50 µl of acetylacetone and incubated at 100°C for 15 min. The solution was then cooled to room temperature, and 3.5 ml of Ehrlich's reagent was added, followed by a 15 min rest. The absorbance of this solution was measured at a wavelength of 556 nm for 20 min using a Double Beam U-2900 Spectrophotometer. Finally, the concentration of 5-ALA was calculated using a standard curve of the 5-ALA reference standard with concentrations ranging from 0 to 30 µg ml − 1 . Crop management practices The crop management practices used in this study included fertilizer application, irrigation frequency, weeding, and pest and disease management. For dry field preparation, a basal application of farmyard manure was used. Throughout plant growth, the "Heiwangte No. 43" compound fertilizer 15-15-15-3(MgO) 50(O.M.), purchased from Taiwan Fertilizer Co., Ltd., was applied in three splits at the early leaf development stage, mid-tillering stage, and early reproductive stage. This application schedule was based on the recommendations of the Miaoli District Agricultural Research and Extension Station, Miaoli County, Taiwan, R.O.C. The wet and dry technique was employed to irrigate the rice crop field. This involved irrigating the field for 24 hours and leaving it to dry for three days. This method ensured that the plant roots had access to enough oxygen to carry out respiration while also reducing weed and algae growth. If weeds were still present in the field, manual weeding methods, such as drowning the weeds in mud, were used. For algae growth, Bacillus subtilis was used in addition to the wet and dry method to control the remaining algae. The dead algae also served as a nitrogen source for rice crop plants. Pest and disease control for the rice crop was achieved using organic pesticides, sprayed fortnightly after PNSB treatment was applied. The organic pesticides used were a mixture of saponin, 50% phosphorous acid, and 50% potassium hydroxide. To control snail populations in the early stages of plant growth, organic tea seed cake pallets (16% saponin), an extract from camellia seeds, were spread across the field. Field data collection From WAT 4, field data were collected to evaluate the growth and development of the rice crop. This included plant height measurements, tiller number, leaf chlorophyll content, and plant lodging resistance. Ten random plants were selected and marked for fixed data collection weekly until WAT 12 (early reproductive or heading stage) to ensure accuracy in data collection. Prior to data collection, the field was partly dried to ensure accuracy in measurements of plant height and tiller number. Plant height was measured using a simple measuring tape, while individual tillers were carefully counted. For leaf chlorophyll content, the SPAD-502 chlorophyll meter (Konica Minolta, Inc., Japan) was used to determine the relative amount of chlorophyll in the rice crop leaf. Relative chlorophyll content was analyzed at six points on each of the three selected leaves from each plant. Plant lodging resistance was determined using the YYD-IB Plant Stem Strength Tester (Wenzhou Tripod Instrument Manufacturing Co., Ltd., China). Additionally, yield and yield-related traits were evaluated following crop harvest. Statistical analysis Data was collected and recorded in Microsoft Excel® 365 (Microsoft Corporation, Washington, DC, USA). Statistical analysis was performed using International Business Machines SPSS Statistics for Windows, version 26 (International Business Machines Corporation, Armonk, New York, USA), with mean comparison conducted using one-way analysis of variance (ANOVA) and mean separation performed using Duncan's multiple-range test. The results were presented as mean ± standard error. Graphs and charts were created using Origin 2019b software (Origin Lab Corporation, Northampton, USA). Results Growing environment conditions The environmental conditions were extensively monitored in both trials, encompassing a comprehensive range of factors, such as air temperature, relative humidity, light intensity, and duration of sunshine hours. The results revealed similar air temperature patterns in both trials, with minimal variations (Fig. 3 a). Notably, the second trial exhibited significantly higher temperatures at WAT 1, WAT 4, and WAT 5 compared to the first trial. Conversely, the first trial showed a significantly higher temperature at WAT 8 than the second. Despite these differences, the maximum temperature remained consistent between the trials, around 30.3°C and 30.7°C for the first and second trials, respectively. Similarly, the minimum temperature showed slight variation, with 19.6°C in the first trial and 18.8°C in the second. Rice cultivation thrives within an optimal temperature range of 25–35°C (Hussain et al. 2019 ). The minimum temperature that rice can tolerate is 15°C (Chand 2014 ). Deviations from this range, either lower or higher, adversely impact crop growth, physiological processes, and, ultimately, yield. In the current study, the data shows a stable and consistent thermal environment for the experimental conditions, with minor variations at specific time points. The relative humidity data showed significant variations between the trials (Fig. 3 b), with the first trial exhibiting significantly higher than the second, except for specific time points (WAT 13, WAT 14, WAT 15, WAT 16, WAT 18, and WAT 20). The first trial had a higher maximum relative humidity of 85.6% compared to the second trial, which averaged around 76.3%. The minimum relative humidity also varied, with the first trial recording approximately 63.4%, while the second trial had 54.5%. The comparison of relative humidity between the first and second trials highlights significant variations in humidity levels, indicating the influence of environmental conditions on rice growth and development. However, the ideal relative humidity for rice cultivation typically ranges between 60% and 85% (Rathnayake et al. 2016 ). In our study, the relative humidity levels for both trials remained within this optimal range, ensuring suitable conditions for the growth and development of the crop. Moreover, the analysis of light intensity also revealed no significant variations between the first and second trials (Fig. 4 a). The maximum light intensity was approximately 692 µmol m − 2 s − 1 at WAT 9 in the first trial. In contrast, in the second trial, the maximum light intensity recorded was 688.2 µmol m − 2 s − 1 at WAT 11. On the other hand, the minimum light intensity in the first trial was 306 µmol m − 2 s − 1 at WAT 18 and 332 µmol m − 2 s − 1 at WAT 18. Previous studies have shown that the ambient light intensity for rice cultivation typically ranges between 300–500 µmol m − 2 s − 1, with ± 100 µmol m − 2 s − 1 considered acceptable (Huang et al. 2013 ). Light intensity above this range can adversely affect rice productivity. Fortunately, the light intensity for both trials in our study fell well within the specified range, ensuring favorable rice crop growth and development conditions. Regarding sunshine hours, the second trial exhibited significantly higher values from WAT 1 to WAT 11, coinciding with crucial stages of plant growth (Fig. 4 b). Both trials reached a maximum of 14 hours of sunshine, particularly during the later stages of the experimental period. However, there was a slight disparity in the minimum sunshine hours, with the first trial averaging approximately 8.6 hours and the second trial averaging around 11 hours. Rice plants display optimal growth when exposed to 12–14 hours of daily sunlight (Jagdish 2023 ). In our study, the sunshine hours were consistently within this range, with slight variations. This consistent exposure to the appropriate amount of sunlight provides favorable conditions for the rice plants to thrive and achieve their full growth potential. Soil nutrient change The soil nutrient change was measured after each trial, and the results are presented in Fig. 5 . Interestingly, individual applications of PNSB and crop rotation demonstrated improvements in soil fertility, but when these treatments were combined, the effect was even more pronounced. Soil pH plays a crucial role in nutrient availability for plants, and the pH range of 5-7.5 is considered optimal for rice cultivation, although rice plants can tolerate a wide range of soil pH levels. In this study, the soil pH remained within the suitable range (Fig. 5 a). Previous research has suggested that crop rotation can lead to an increase in soil pH (Ouda et al. 2018 ; Li et al. 2023 ). However, contrary to those findings, the current study observed a significant drop in soil pH with crop rotation, and this decline was more pronounced when combined with PNSB inoculation. This discrepancy could be attributed to the unique crop species chosen for rotation in this study, leading to different effects on soil pH compared to previous research (Ouda et al. 2018 ). Moreover, although not statistically significant, crop rotation positively impacted the soil organic matter (OM) in both fields, which declined in the control field after rice cultivation (Fig. 5 a). Conversely, in the PNSB-inoculated field, the bacteria contributed to maintaining soil OM levels. A similar trend was observed for soil P levels, where crop rotation initially increased P concentration in both fields, but later, it declined in both (Fig. 5 b). However, the rate of decline in the PNSB-inoculated field was higher, possibly due to PNSB's role in making soil P more available for plant uptake by solubilizing it (Khuong et al. 2018 ; Huu et al. 2022 ). Similarly, crop rotation led to enhanced soil K and Ca levels in both fields, but in the PNSB-treated field, these nutrients experienced further enhancement, while there was a decline in the control field, indicating the synergistic effects of crop rotation and PNSB inoculation. Furthermore, in the control field, with crop rotation, the Mg, Fe, Mn, and Zn concentrations either remained unchanged or decreased (Fig. 5 ). In contrast, the PNSB inoculated field showed a slight decline in Mn and Zn concentration, with Mg and Fe levels remaining relatively constant. However, the combined treatment resulted in a significantly higher increase in these nutrients compared to the control field, which exhibited only a slight increase. Notably, Cu concentration increased with crop rotation in both fields, with a significant increase in the control field and a significant decline in the PNSB field. Meanwhile, Na concentration declined in both fields after crop rotation but recovered with rice cultivation (Fig. 5 a). Overall, the findings highlight the highly effective synergetic effects of crop rotation and PNSB inoculation in enhancing soil nutrient levels. Antioxidant enzyme activity The study analyzed the activity of several antioxidant enzymes, including SOD, APX, CAT, and GR, to determine whether the plants were exposed to stress. The results indicated that PNSB inoculation positively impacted SOD, APX, and GR activity, with decreases of 5%, 3%, and 13%, respectively, as shown in Table 1 . However, there was an increase in CAT activity in the control field compared to the PNSB-inoculated field. Despite some fluctuations in antioxidant enzyme activity during the experiment, the changes observed were not significant, as shown in Table 1 . These results indicate that the plants were not subjected to any forms of stress that could negatively impact their growth performance and yield. The application of PNSB was found to be beneficial and well-received by the crop. Table 1 Antioxidant enzyme activity of rice plants under field conditions with and without PNSB treatment Treatments Superoxide dismutase (SOD) Ascorbate peroxidase (APX) Catalase (CAT) Glutathione reductase (GR) units mg − 1 protein Control 0.139 ± 0.010 a 0.092 ± 0.008 a 0.015 ± 0.001 a 0.016 ± 0.010 a PNSB 0.132 ± 0.012 a 0.089 ± 0.007 a 0.018 ± 0.002 a 0.014 ± 0.009 a Values are mean ± SE (n = 5). Means in the same column, followed by the same letter(s), are not significantly different (p ≤ 0.05) based on Duncan's multiple range test. Below-ground environment conditions In the second rice cultivation, below-ground environmental conditions were assessed as indicators of plant stress, focusing on soil temperature and soil EC. The results revealed no significant differences in soil temperature between the control and treatment fields, except at WAT 10, WAT 11, WAT 12, and WAT 18 (Fig. 6 a), with maximum temperatures of 26.6°C and 26.3°C in the control and treatment fields, respectively. Similarly, the minimum temperatures were 19.3°C and 19.5°C for the control and treatment fields, respectively, indicating that the temperatures in both fields were maintained within the optimal range of 19.0°C to 27.0°C. Conversely, soil EC was significantly higher in the treatment field compared to the control field (Fig. 6 b), with maximum EC values of 0.43 dS/m and 0.57 dS/m in the control and treatment fields, respectively. The minimum EC values were 0.13 dS/m and 0.25 dS/m in the control and treatment fields, respectively. Studies have shown that the optimum temperature of 25°C for root growth (Arai-Sanoh et al. 2010 ) and EC levels below 4 dS/m (Ding et al. 2018 ) is suitable for the healthy growth of rice with a higher yield. Although there were slight variations, the soil temperature and EC levels were within this specified range in the current study (Fig. 6 ). Analysis of 5-aminolevulinic acid The application of PNSB had notable effects on the 5-ALA content in rice crop plants at different time points, as shown in Fig. 7 . At WAT 4, a slight difference in 5-ALA concentration was observed between the control and PNSB-treated groups, which was not significant. However, at WAT 9, a significant 12% increase in 5-ALA content was evident in the PNSB-treated plants compared to the control group. This trend continued at WAT 14, with the PNSB-treated group showing a significant 10% highest 5-ALA concentration. These results suggest that PNSB application may positively influence the synthesis or accumulation of 5-ALA in rice crop plants, particularly at later stages of growth as the bacterial population increases. The 5-ALA is a common precursor of tetrapyrroles and is vital as a growth regulator in higher plants (Senge et al. 2014 ; Wu et al. 2018 , 2019 ). Studies have demonstrated the effectiveness of 5-ALA in enhancing photosynthesis and mitigating the detrimental impacts of diverse abiotic stresses on higher plants. In our present study, the application of PNSB resulted in an increase in 5-ALA in plants. This elevation of 5-ALA strengthened the plants' resilience to abiotic stress and substantially improved their overall growth and yield. Above-ground plant performance Plant height is a crucial agronomic characteristic that significantly impacts rice yield potential (Zhang et al. 2017 ; Li et al. 2019 ). While the dwarf phenotype offers advantages in preventing lodging, excessively short plants can result in inadequate growth, ultimately hampering the overall yield potential of rice (Zhang et al. 2017 ). Striking the right balance in plant height is essential for optimizing rice production and maximizing crop productivity. According to the Kaohsiung District Agricultural Research and Extension Services, the average plant height of the Kaohsiung 147 rice variety is around 98.1 cm in the main growing season. The average plant height for control was 95.4 cm; however, the results of this study show that PNSB inoculation led to a non-significant 4% increase in plant height (Fig. 8 a). When PNSB inoculation was combined with crop rotation, there was a significant 9% increase in plant height during the second year compared to crop rotation alone (Fig. 8 b). The notable increase in plant height with PNSB treatment is likely attributed to the enhanced availability of soil nutrients (Ye et al. 2019 ; Shankar et al. 2022 ) facilitated by the synergistic effects of crop rotation and PNSB inoculation, as depicted in Fig. 5 . This study presents compelling evidence that the simultaneous implementation of crop rotation and PNSB inoculation yielded positive effects on the growth performance of rice plants. Additionally, the one-way ANOVA revealed that the overall plant height in the first year was significantly higher than in the second year, as depicted in Fig. 8 . These plant height variations could be attributed to differences in sunshine duration. Reduced sunshine hours may result in increased plant height, whereas longer exposure to sunlight might keep plants shorter, as observed in this study (Fig. 4 b and Fig. 8 ). Therefore, the right balance in the availability of nutrients in the soil and to plants with the right growth environment allows plants to focus more on grain yield rather than vegetative growth, which was evident in this study. Striking this balance proved critical, allowing the plants to channel their energy and resources more efficiently toward producing higher grain yields. Moreover, the study demonstrated that treating rice crops with PNSB and crop rotation significantly affected the leaf chlorophyll level, which is important for plant photosynthesis. The application of PNSB resulted in a significant 4% increase in leaf chlorophyll levels compared to the control group (Fig. 9 a). On the other hand, the combined treatment of PNSB and crop rotation led to a significant 3% increase in leaf chlorophyll levels compared to crop rotation alone (Fig. 9 b). Crop rotation alone and the combined treatment of crop rotation and PNSB significantly increased the leaf chlorophyll level by 9% compared to the control and PNSB inoculation, respectively (Fig. 9 ). The one-way ANOVA results suggest that although PNSB inoculation alone can increase leaf chlorophyll levels; however, combining it with crop rotation as treatment can lead to a significantly greater increase in chlorophyll levels. The study unequivocally demonstrates the substantial advantage of crop rotation on leaf chlorophyll concentration. Remarkably, when combined with PNSB inoculation, a noteworthy additional increase in leaf chlorophyll levels is observed. This synergistic effect can be attributed to the improved nutrient availability in the soil (Fig. 5 ), which plays a pivotal role in enhancing plant growth. Adequate nutrient supply boosts leaf chlorophyll levels, a crucial component for efficient photosynthesis. These findings underscore the significance of nutrient management strategies, particularly in the context of combined agricultural practices, and shed light on the mechanisms behind the observed enhancements in plant photosynthetic capacity. Furthermore, the tiller number in rice crop is also one of the key indicators of grain yield (Li et al. 2003 ; Zhao et al. 2020 ). In the current study, both PNSB inoculation and crop rotation positively affected the number of tillers in rice plants. Specifically, PNSB inoculation resulted in a 13% increase in the tiller number compared to the control group, although it was not statistically significant (Fig. 10 a). On the other hand, crop rotation led to a significant 107% increase in the tiller number compared to the control group (Fig. 10 ). When PNSB inoculation was combined with crop rotation, a significant 27% increase in tiller number was observed compared to crop rotation alone (Fig. 10 b), and a significant 133% increase compared to PNSB inoculation alone (Fig. 10 ). The one-way ANOVA suggests that PNSB inoculation and crop rotation are effective strategies for enhancing the tiller number of rice plants. In particular, crop rotation can substantially increase tiller number, while PNSB inoculation can provide a significant but relatively smaller increase. However, combining PNSB inoculation and crop rotation may offer the most favorable outcomes for enhancing the tiller number in rice cultivation. The significant increase in tiller number could be a result of the enhanced soil nutrient (Tian et al. 2017 ; Zha et al. 2022 ; Zhou et al. 2022 ; Shankar et al. 2022 ) by the combined effects of crop rotation and PNSB inoculation (Fig. 5 ). The observed increase in tiller number directly correlates with higher grain yield (Koprna et al. 2021 ; Yen et al. 2022 ), assuming no interference from other biotic or abiotic factors that might impact the crop growth. Finally, crop lodging is one of the limiting factors in rice production, and enhancing the lodging resistance of plants can ensure higher grain yield (Zhang et al. 2014 ; Liu et al. 2018 ; Shah et al. 2019 ; Guo et al. 2021 ; Luo et al. 2022 ; Tsugawa et al. 2023 ). In this study, both PNSB inoculation and crop rotation significantly increased the lodging resistance of rice plants. The PNSB inoculation resulted in a remarkable 44% increase in lodging resistance compared to the control (Fig. 11 ). In comparison, the crop rotation led to a significant 27% increase compared to the control (Fig. 11 ). Moreover, combining the two treatments resulted in a significant 31% increase in lodging resistance compared to crop rotation alone and a significant 16% increase compared to PNSB inoculation alone (Fig. 11 ). The one-way ANOVA analysis shows that both PNSB inoculation and crop rotation can effectively enhance the lodging resistance of rice plants. Though PNSB inoculation may improve lodging resistance more than crop rotation, combining them may offer a greater result in rice cultivation. The observed improvement in lodging resistance in the rice crop can be attributed to the synergistic effects of crop rotation and PNSB inoculation, which led to significant increases in both root growth and tiller numbers. The combination of longer roots, higher tiller numbers, and shorter plants creates favorable conditions to prevent lodging in rice crop plants. This optimal plant structure enhances stability and resilience against bending or collapsing due to wind or heavy rain, thus minimizing yield losses. Consequently, these improvements contribute to increased harvestable yield and overall grain quality. Therefore, this study proves that these combined agricultural practices work harmoniously to enhance the rice plants' overall structural integrity, making them better equipped to withstand lodging and ultimately contributing to more robust and stable crop yields. Below-ground plant performance The current study also investigated the effects of PNSB inoculation on the below-ground performance of rice. The results demonstrate that PNSB inoculation significantly enhanced the root growth of rice plants, as shown in Table 2 . Specifically, PNSB inoculation resulted in a 33% increase in root length and a 130% increase in root dry weight compared to the control plants. Although there was a 67% increase in root volume with PNSB inoculation, this increase was not statistically significant. These findings indicate that PNSB inoculation can significantly improve the below-ground performance of rice plants, particularly in terms of root length and dry weight. This could be due to the increase in 5-ALA concentration (An et al. 2019 ; Ma et al. 2022 ), a precursor of tetrapyrroles produced through PNSB inoculation. As a consequence of these enhancements, the rice plants' nutrient uptake capacity is likely to be augmented. With more robust and extensive root systems, plants can access a larger volume of soil, allowing them to absorb essential nutrients more efficiently. This improved nutrient uptake, combined with the growth-promoting effects of PNSB, ultimately leads to a substantial enhancement in the overall growth and productivity of rice crops. Table 2 Root growth performance of rice plants inoculated with PNSB compared to uninoculated control in the first trial Treatments Root length (cm) Root volume (cm 3 ) Root dry weight (g) Control 43.5 ± 0.21 b 200 ± 0.00 a 18.1 ± 1.20 b PNSB 57.8 ± 1.65 a 333 ± 1.11 a 41.6 ± 2.01 a Values are mean ± SE (n = 3). Means in the same column, followed by the same letter(s), are not significantly different (p ≤ 0.05) based on Duncan's multiple range test. Yield components and grain metrics The yield-related characteristics of rice, including productive tillers per hill, average grain per hill, grain fertility, and 1000-grain weight, were comprehensively evaluated (Table 3 ). The results unveiled the significant impact of PNSB application on rice crop plants. Specifically, including PNSB led to a significant enhancement of 34% in productive tillers per hill, showcasing its positive influence on tiller formation. Moreover, the implementation of crop rotation resulted in a remarkable 94% increase in productive tillers per hill compared to untreated plants, highlighting the effectiveness of this practice in promoting tiller development. However, when PNSB and crop rotation were combined, the observed increase of 2% in productive tillers per hill was not statistically significant. While the combined effect did not significantly contribute to additional tiller formation compared to crop rotation alone, it is essential to highlight that the synergy between PNSB and crop rotation still yielded superior results compared to PNSB inoculation alone. This integration led to a noteworthy increase of 48% in productive tillers per hill. The outcome underscores the positive impact of the crop rotation and PNSB combination, suggesting a more favorable growing environment and enhanced nutrient supply, likely attributed to the presence of shredded djulis stem. Table 3 Yield characteristics and grain metrics of rice under different treatment conditions Parameters Year 1 Year 2 Control PNSB CR CR + PNSB Productive tillers/ hill (%) 49.6 ± 2.56 c 66.3 ± 2.97 b 96.4 ± 1.83 a 98.0 ± 0.65 a Average grain/ hill (g) 22.0 ± 1.46 c 33.7 ± 3.06 b 40.2 ± 1.77 a 45.3 ± 2.16 a Grain fertility (%) 70.1 ± 3.57 c 78.9 ± 1.57 b 96.3 ± 0.57 a 96.0 ± 0.33 a 1000 grain weight (g) 23.9 ± 0.16 a 24.4 ± 0.11 a 20.8 ± 0.33 b 21.4 ± 0.29 b Values are mean ± SE (n = 8). Means in the same row, followed by the same letter(s), are not significantly different (p ≤ 0.05) based on Duncan's multiple range test. The average grain per hill analysis revealed substantial effects of the various treatments (Table 3 ). The inoculation of PNSB significantly increased the average grain per hill by 53%, highlighting its positive influence on grain production. Similarly, crop rotation alone resulted in a significant 83% increase in average grain per hill, emphasizing the efficacy of this practice in enhancing grain yield. When PNSB was combined with crop rotation, there was a 13% increase in average grain per hill compared to crop rotation alone. Although this increase was not statistically significant, combining both approaches suggests a potential synergistic effect. Importantly, when comparing the combination of PNSB with crop rotation to PNSB inoculation alone, a significant 34% increase in average grain per hill was observed, further highlighting the added benefit of combining these treatments. The analysis of grain fertility revealed significant effects of the different treatments (Table 3 ). According to Kaohsiung District Agricultural Research and Extension Station ( 2023 ), the grain fertility of the Kaohsiung 147 rice variety is around 68.2% in the main season. However, in this study, grain fertility was around 70.1% in the control field, which was 3% higher than recommended. Conversely, the inoculation of PNSB led to a notable 13% improvement in grain fertility, highlighting its positive impact on this important yield attribute. Additionally, crop rotation alone resulted in a substantial 38% increase in grain fertility, emphasizing its effectiveness in enhancing the reproductive capacity of rice plants. Interestingly, when comparing crop rotation alone to the combination of crop rotation with PNSB inoculation, a slight variation of approximately 0.30% was observed in grain fertility. Although not statistically significant, this observation suggests a potential synergistic effect between the two treatments, indicating that they may complement each other in enhancing grain fertility. Furthermore, when PNSB was combined with crop rotation, a significant 22% increase in grain fertility was observed compared to PNSB inoculation alone. This finding highlights the positive interaction between PNSB and crop rotation, indicating that their combined application can lead to even greater improvements in grain fertility. Moreover, the analysis of 1000-grain weight yielded intriguing results (Table 3 ). According to Kaohsiung District Agricultural Research and Extension Station ( 2023 ), the average 1000-grain weight of the Kaohsiung 147 rice variety is around 23.6 g in the primary growing season. However, in our study, the 1000-grain weight in the control field was around 23.9 g, which was 1% higher than the recommended. Conversely, the inoculation of PNSB led to a modest 2% increase in grain weight compared to the control, but this difference was not statistically significant. In contrast, crop rotation alone showed a significant 13% decrease in grain weight. Likewise, when PNSB inoculation was combined with crop rotation, a significant 12% decrease in grain weight was observed compared to PNSB inoculation alone. Interestingly, comparing the combination of PNSB inoculation with crop rotation to crop rotation alone showed a slight 3% increase in grain weight, although not statistically significant. This result indicates that pursuing higher grain yield may come at the expense of a lower 1000-grain weight. However, there is potential for improvement by implementing additional soil nutrient enhancements. Grain yield and resource allocation The evaluation of the final yield encompassed key parameters such as grain yield, shoot dry weight, and harvest index (Table 4 ). According to Kaohsiung District Agricultural Research and Extension Station ( 2023 ), the grain yield of Kaohsiung 147 rice variety in the main season is around 6.18 tonnes per hectare. However, our study revealed that the grain yield in the control field was approximately 5.54 tonnes per hectare, which was 12% lower than the recommended yield. This discrepancy could be attributed to long-term monocropping practices, resulting in reduced soil fertility and a less favorable growing environment. Nonetheless, the inoculation of PNSB demonstrated a substantial impact, leading to a 61% significant increase in grain yield. Similarly, crop rotation showed a 71% significant increase in grain yield. Remarkably, when PNSB was combined with crop rotation, a significant 19% increase in grain yield was observed compared to crop rotation alone. Furthermore, the combined effects of PNSB and crop rotation resulted in a 27% significant increase in grain yield compared to PNSB inoculation alone. The results highlight that implementing crop rotation or PNSB inoculation individually can lead to higher grain yields in rice, likely attributed to improved soil fertility. Nevertheless, the synergy achieved by combining and applying both treatments demonstrates an even more substantial increase in grain yield, showcasing the potential for enhanced agricultural productivity. Table 4 Grain yield and resource allocation in rice crop plants under different treatment conditions Parameters Year 1 Year 2 Control PNSB CR CR + PNSB Grain yield (t ha − 1 ) 5.54 ± 0.43 c 8.93 ± 0.60 b 9.45 ± 0.48 b 11.3 ± 0.51 a Shoot dry weight (t ha − 1 ) 21.8 ± 1.72 b 23.8 ± 1.54 b 22.2 ± 0.53 b 27.9 ± 0.56 a Harvest index 0.26 ± 0.02 c 0.38 ± 0.01 b 0.42 ± 0.01 a 0.40 ± 0.02 ab Values are mean ± SE (n = 8). Means in the same row, followed by the same letter(s), are not significantly different (p ≤ 0.05) based on Duncan's multiple range test. Moreover, the shoot dry weight with PNSB inoculation contributed to a notable 9% increase, even though it was not statistically significant (Table 4 ). Similarly, crop rotation exhibited a modest 2% increase in shoot dry weight. However, when PNSB was combined with crop rotation, a significant 26% increase in shoot dry weight was observed compared to crop rotation alone. Furthermore, the combined effects of PNSB and crop rotation yielded a 17% significant increase in shoot dry weight compared to PNSB inoculation alone. These results demonstrate that the combined application of PNSB with crop rotation positively influences shoot dry weight, showcasing a synergistic effect that contributes to improved plant growth and productivity. Finally, PNSB inoculation resulted in a substantial 46% increase in the harvest index (Table 4 ). Similarly, crop rotation showed a 62% significant increase in the harvest index. However, when PNSB was combined with crop rotation, a slight 5% decrease in the harvest index was observed compared to crop rotation alone, which was not a significant decrease. Nevertheless, PNSB and crop rotation combined effects demonstrated a significant 5% increase in the harvest index compared to PNSB inoculation alone. These findings underscore the potential of PNSB inoculation and crop rotation as effective strategies for enhancing rice yield and growth parameters, including grain quality. Discussion Growing environment conditions Environmental conditions play a crucial role in the growth and development of rice. Maintaining optimal conditions, including appropriate air temperature, relative humidity, light intensity, and sufficient sunshine hours, promotes healthy rice growth and maximizes crop yield. Rice cultivation requires precise air temperature conditions, and the ideal range for successful growth and development is typically between 25 and 30°C (de los Reyes et al. 2003 ). Photosynthesis, a vital process for plant growth, exhibits an optimal air temperature range of 14 to 32°C (Parent et al. 2010 ). Deviations beyond this range can negatively impact photosynthetic efficiency. In our study, the air temperature in both trials ranged from 19 to 31°C (Fig. 3 a). Although it was slightly outside the optimum range for rice cultivation, it fell within the range for optimal photosynthesis. Relative humidity also holds significant importance in rice cultivation, impacting yield (Yan et al. 2010 ). The ideal relative humidity for rice cultivation typically ranges between 60% and 85% (Rathnayake et al. 2016 ). In our study, the relative humidity ranged from 54–85% (Fig. 3 b) in both trials, falling within the suitable range. The combined influence of temperature and relative humidity plays a significant role in rice cultivation, acting as the predominant controlling factors, given their spatial and temporal variability (Rathnayake et al. 2016 ). Moreover, light intensity and duration of sunshine hours are closely intertwined and directly influence rice yield and quality (Liu et al. 2014 ; Shafiq et al. 2021 ). Rice plants exhibit optimal growth when exposed to 12–14 hours of daily light (Jagdish 2023 ), with ambient light intensity typically ranging between 300–500 µmol m − 2 s − 1, with ± 100 µmol m − 2 s − 1 considered acceptable (Huang et al. 2013 ). In the current study, the light intensity ranged from 306 to 692 µmol m − 2 s − 1 (Fig. 4 a) for both trials, slightly deviating from the ideal range but mostly within acceptable limits. Similarly, the average daily sunshine hours were around 12 hours (Fig. 4 b) in both trials, aligning with the optimum requirements for rice growth. On the other hand, in the first trial, we analyzed antioxidant enzyme activity to assess whether the plants experienced any stress during the growth period, potentially influencing the results. Surprisingly, the results revealed no significant differences in antioxidant enzyme activity between the control and treatment fields, indicating that both sets of plants were not subjected to any noticeable forms of stress (Table 1 ). In the second trial, a different approach was taken to assess potential plant stress conditions. Instead of analyzing antioxidant enzyme activity, we focused on the below-ground environment by examining soil temperature and EC levels. This allowed us to gain insights into the influence of below-ground factors on rice growth. In addition to above-ground conditions, factors such as soil temperature and EC levels are key determinants of rice growth and development. Previous studies have indicated that soil temperature significantly affects both grain yield and quality, with rice plants showing optimal root development at around 25 ºC (Arai-Sanoh et al. 2010 ). In our study, the average soil temperature in both control and treatment fields was approximately 24 ºC (Fig. 6 a), closely approaching the optimal range. Furthermore, soil EC is of utmost importance for successful rice cultivation. It directly influences nutrient availability, water movement, and overall soil health, thereby impacting the growth and yield of rice plants. Maintaining an appropriate soil EC level is crucial for optimal nutrient uptake, root development, and overall plant vigor (Ding et al. 2018 ). For higher yield, it is recommended to cultivate rice, along with most crops, in low to no-saline soils with an EC below 4 dS/m. In our study, the soil EC levels ranged from 0.13 to 0.57 dS/m in both control and treatment fields, with the treatment field showing significantly higher EC levels (Fig. 6 b). Soil nutrient change The combination of crop rotation with PNSB demonstrated remarkable effects on soil nutrient enrichment, as evident from the soil nutrient analysis results (Fig. 5 ). Crop rotation has long been recognized for its ability to improve soil nutrients, as supported by various studies (Francis 2005 ; Karlen et al. 2006 ; Asseng et al. 2014 ; Dhaliwal et al. 2021 ; Liu et al. 2022 ; Zani et al. 2022 ). To achieve this, selecting appropriate crops that can fix N or enhance nutrient availability is vital. Legumes, for instance, play a valuable role in adding N to the soil through fixation and can be used as green manure (Rangarajan 2009 ). In the present study, djulis, although not an N-fixing crop like legumes, demonstrated its significance by offering an alternative approach. Even though djulis does not fix N, its tissues contain abundant essential nutrients, including K, P, Mg, Ca, Na, Fe, and Zn (Tsai 2022 ). After harvest, the djulis plant material was shredded and spread across the field before rice planting. This practice allowed the nutrients to gradually leach into the soil, at the same time providing a favorable environment for colonizing beneficial bacteria, such as PNSB, which also assists in enhancing soil nutrients. The PNSB then utilizes a specific nitrogenase enzyme to convert atmospheric molecular N into NH 3 or NH4 + , effectively making N accessible for plant uptake (Franche et al. 2009 ; Olivares et al. 2013 ). The PNSB also has the ability to improve plant nutrition by mobilizing and increasing the availability of nutrients (Lee et al. 2021 ). For example, the elite R. palustris strain TN110 was found to possess three sets of Mo, V, and Fe nitrogenase gene clusters. As a consequence of this genetic makeup, TN110 released notably higher concentrations of NH 4 + compared to the other strains tested in the study (Sakpirom et al. 2017 ). Moreover, PNSB possesses the capability to solubilize P in the soil. In the context of acid sulfate soil, the combination of low pH and high levels of Al, Fe, H 2 S, and organic acids often leads to reduced P availability due to its immobilization with Al 3+ and Fe 2+ . This results in the formation of insoluble compounds like AlPO 4 •H 2 O and FePO 4 •H 2 O (Yadav and Verma 2012 ; Barrow 2017 ; Nguyen et al. 2018 ; Andrino et al. 2021 ; Khuong et al. 2021 ). However, PNSB can counteract this issue by producing organic acids that interact with these soil minerals, liberating bound P in a soluble form (Khuong et al. 2018 , 2021 ). As a result, P becomes readily accessible for absorption by plant roots, leading to enhanced soil quality, improved nutrient uptake, and, ultimately, increased crop yield. This study is very important as it shows the synergistic benefits of combining crop residues and PNSB application, effectively enriching the soil with essential nutrients (Fig. 12 ). Additionally, Fe is an element for plant growth and development, as it is essential in the synthesis of chlorophyll, which plays a vital role in photosynthesis (Rout and Sahoo 2015 ; Kathpalia and Bhatla 2018 ; Tripathi et al. 2018 ; Rai et al. 2021 ; Arif et al. 2022 ). In the present study, the PNSB-treated field showed higher Fe accumulation than the control, indicating an enhanced Fe availability in the soil. The increased Fe availability observed in the soil could be attributed to the presence of siderophores, which are small organic molecules secreted by bacteria with a strong affinity for Fe 3+ (Albelda-Berenguer et al. 2019 ; Kramer et al. 2020 ; Khasheii et al. 2021 ). Siderophores are produced by almost all known bacterial species (Guerinot 1994 ; Ratledge and Dover 2000 ; Miethke and Marahiel 2007 ), including PNSB (Nookongbut et al. 2019 ; Khuong et al. 2020a , b ). In situations where Fe is limited or exists in an insoluble form in the soil, PNSB releases siderophores that form stable complexes by chelating or binding with Fe 3+ . Subsequently, these siderophore-Fe complexes are taken up by PNSB cells, releasing the bound Fe inside the bacterial cells. This efficient process allows PNSB to effectively acquire Fe from the environment, making it available for both the bacteria and the surrounding plants. Synergetic effects on rice growth and yield The inoculation of PNSB exhibited a favorable response toward plant growth, and even more promising outcomes were observed with crop rotation alone. However, the most remarkable results were achieved when both practices were combined. The PNSB can promote plant growth by improving nutrient acquisition, producing plant growth-promoting substances, inducing immune system responses, and interacting with the resident microbial community (Fig. 12 ). In addition to fixing N for plants, PNSB has been reported to improve N use efficiency (NUE) in plants in the presence of N fertilizer (Wong et al. 2014 ; ShuHua et al. 2015; Hsu et al. 2021 ). For example, inoculation of rice with R. capsulatus DSM155, along with N fertilizer, resulted in a significant 20% increase in nitrogen content within the roots (Elbadry and Elbanna 1999 ). However, it is worth noting that the impact of inoculation was comparatively diminished when N fertilizer was present, in contrast to N-deficient conditions. Likewise, PNSB has also been shown to lower the NO 3 − content in crop leaves, which could otherwise have an impact when consumed (Wong et al. 2014 ). Moreover, PNSB also produces plant growth-promoting substances such as 5-ALA, IAA (indole-3-acetic acid, siderophores, and exopolymeric substances (Nookongbut et al. 2019 ). The IAA, an auxin, exerts multiple positive effects on plant growth and development (Vessey 2003 ; Tsavkelova et al. 2006 ; Bending et al. 2007 ; Kaymak 2011 ; Wani et al. 2016 ), including cell division, root initiation, flowering, fruit setting, ripening, senescence, and gravitropism (Talukdar et al. 2022 ). The PNSB has the capability to synthesize IAA using two pathways: indole-3-pyruvate (IPA) and tryptamine (TAM) pathways, both of which utilize tryptophan as a precursor molecule (Spaepen et al. 2007 ; Mujahid et al. 2011). On the other hand, 5-ALA produced by PNSB serves as a significant precursor for tetrapyrrole compounds in plants, including essential components like chlorophyll, heme, and vitamin B12 (Kang et al. 2012 ). Additionally, 5-ALA acts as a typical plant growth regulator (Wu et al. 2018 ), exerting influence over various aspects of plant growth, development, and overall yield (Wang et al. 2021a ). Prior research has demonstrated the positive effects of applying 5-ALA to plants, such as improved photosynthesis (Wu et al. 2018 ), mitigation of abiotic stresses (Wu et al. 2018 , 2019 ; Wang et al. 2021a ), and enhanced fruit quality (Wang et al. 2021a ). Certain photosynthetic bacteria, including specific strains of R. palustris , can synthesize 5-ALA (Sasaki et al. 1987 ). For instance, under NaCl stress conditions, R. palustris strains have been found to secrete approximately 2.67 µM of 5-ALA, thereby promoting the growth and biomass of rice roots (Nunkaew et al. 2014 ). In addition, 5-ALA also plays a significant role in the regulation of chlorophyll in plants. When applied to plants, 5-ALA promotes the availability of protoporphyrin IX, a key precursor for chlorophyll synthesis (Harada et al. 2022 ; Sun et al. 2023 ). As a result, the application of 5-ALA leads to an augmentation in chlorophyll concentration, providing several benefits to plants, including enhanced photosynthetic capacity, increased energy production, and an overall improvement in plant growth. This, in turn, can lead to greater biomass accumulation, increased leaf area, and an overall enhancement of plant performance. Priming plants with 5-ALA has also been shown to boost plant resilience against diverse stresses, encompassing drought, salinity, UV-B radiation, and extreme temperatures. This impact is accomplished by finely adjusting enzyme activities, channel proteins, hormones, signaling molecules, small organic molecules, gene expression, and physiological levels. This regulatory mechanism significantly enhances crucial processes such as photosynthesis, osmoregulation, antioxidant capacity, and N assimilation in plants (Tan et al. 2022 ). The present study reveals a substantial increase in 5-ALA in plants following PNSB inoculation (Fig. 7 ). This rise in 5-ALA could potentially contribute to elevated leaf chlorophyll content (Fig. 9 ). However, it is essential to consider that other nutrients like Mg and Fe also play crucial roles in enhancing chlorophyll synthesis in plants (Farhat et al. 2016 ). Consequently, the study's findings demonstrate the synergistic effect of PNSB inoculation and crop rotation, leading to the most favorable overall plant growth performance. Furthermore, the combined effects of crop rotation and PNSB inoculation, which significantly enhanced rice growth, resulted in a remarkable improvement in yield. Specifically, the integration of crop rotation with PNSB led to an impressive 104% increase in grain yield and a substantial 28% increase in shoot dry weight (Table 4 ). As a result, the harvest index rose to an outstanding 54%, reflecting the successful synergy between these two agricultural practices in maximizing rice productivity. Conclusion In conclusion, this research highlights the significant potential of utilizing PNSB in conjunction with crop rotation to foster a synergetic effect on agricultural productivity. The combined approach of PNSB inoculation and crop rotation exhibited a remarkable improvement in soil fertility, as evidenced by enhanced nutrient availability and nutrient cycling processes. Additionally, the presence of PNSB led to a substantial increase in the concentration of 5-ALA in plants, indicating a positive impact on photosynthetic activity and energy production. The observed enhancements in growth parameters, including tiller numbers, leaf chlorophyll content, and lodging resistance, clearly demonstrated the beneficial effects of this integrated approach on rice plants. The positive impact on productive tillers per hill, average grain per hill, and grain fertility ultimately resulted in a significant increase in grain yield and shoot dry weight. The improved harvest index indicates an efficient resource allocation within the crop. These findings suggest that combining PNSB inoculation with crop rotation has promising implications for sustainable agriculture, offering an innovative strategy to address food security challenges. The insights gained from this research could stimulate further investigations in the field of agricultural science and foster the adoption of these novel integrated practices to optimize crop productivity and ensure a more resilient agricultural future. Declarations Acknowledgments: The authors would like to extend their sincere appreciation to the Food Industry Research and Development Institute (FIRDI), situated at 331 Shih-Pin Road, Hsinchu, 300 Taiwan (R.O.C.), for generously supplying the photosynthetic bacteria (Research number PSB32) utilized in this research. Funding: The authors did not receive support from any organization for the submitted work. Authors' contributions: Conceptualization: Laurence Shiva Sundar, Kuei-Shan Yen, Yun-Yang Chao; Methodology: Laurence Shiva Sundar, Kuei-Shan Yen, Yun-Yang Chao; Formal analysis and investigation: Laurence Shiva Sundar, Kuei-Shan Yen, Yao-Tsung Chang; Writing—original draft preparation: Laurence Shiva Sundar; Writing—review and editing: Yun-Yang Chao; Funding acquisition: Yun-Yang Chao; Resources: Yao-Tsung Chang, Yun-Yang Chao; Supervision: Yun-Yang Chao Availability of data and material: Not applicable Competing interests: The authors have no relevant financial or non-financial interests to disclose. References Adelana A, Aduramigba-Modupe V, Oke A, et al (2022) Soil quality assessment under different long-term rice-based cropping systems in a tropical dry savanna ecology of northern Nigeria. 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Plant Production Science 25:311–319. https://doi.org/10.1080/1343943X.2022.2061538 Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies 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-3383462","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":239285276,"identity":"d230e327-be1b-4c2f-bef3-a1efb91da68a","order_by":0,"name":"Laurence Shiva Sundar","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Laurence","middleName":"Shiva","lastName":"Sundar","suffix":""},{"id":239285277,"identity":"950ec611-c4f1-4152-b695-9645092f1e74","order_by":1,"name":"Kuei-Shan Yen","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kuei-Shan","middleName":"","lastName":"Yen","suffix":""},{"id":239285278,"identity":"6f909151-1db1-42f8-9e13-3629561c72da","order_by":2,"name":"Yao-Tsung Chang","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yao-Tsung","middleName":"","lastName":"Chang","suffix":""},{"id":239285279,"identity":"884a8cb6-6cba-4ab3-9026-26b002045e80","order_by":3,"name":"Yun-Yang Chao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYDACCTBpw8DAzNyAJohfSxpQCyNECw9E0ICQlsNATKwW/tnNDx/ztp2P5m9nbGAu3HHY3p6B+eBtHoY/iQ04tEjcOWZszNt2O3fGYaCWmWcOJ/YwsCVb8zAY4NRiIJFgJg3S0gDSwtt2OIGHgcdMGqglF7eW9G9ALedy50O12PMw8H8joCUHZMuB3A1QLYw9DDxseLVI3MgpNpxzLjl3I1DLYd4z6Yk9h9mMLecYGNfj0sI/I33jgzdldrnzzh8++Jh3h7U9e3vzwxtvKuSMcegAAyZeNgjjADhqmMEOxqcBGIc//sBYuBwzCkbBKBgFIxoAANrPUij/Vn4qAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-6642-7205","institution":"National Pingtung University of Science and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yun-Yang","middleName":"","lastName":"Chao","suffix":""}],"badges":[],"createdAt":"2023-09-25 06:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3383462/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3383462/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":44606292,"identity":"8ea72838-646f-4b36-80e2-3d7a45c2e397","added_by":"auto","created_at":"2023-10-13 22:28:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1621695,"visible":true,"origin":"","legend":"\u003cp\u003eUtilization of processed djulis plant materials integrated into the soil prior to rice cultivation to augment soil fertility and serve as conducive substrates for PNSB colonization. (a) Shredded djulis stems and (b) crushed stems uniformly dispersed across the field for implementation\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3383462/v1/32f1d3b0426e93f2d9aec584.png"},{"id":44607938,"identity":"23b0cf9d-993f-4b2c-954f-97883bb4d034","added_by":"auto","created_at":"2023-10-13 22:44:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":697880,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal variation in bacterial culture media coloration over 14 days of cultivation. The numbers on the image represent the respective culture days\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3383462/v1/ccb31bb401a2341c58273ffc.png"},{"id":44793019,"identity":"4b3d519d-f15b-4b12-bd22-b3fde4e876c5","added_by":"auto","created_at":"2023-10-17 14:56:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27288,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of above-ground environmental conditions between the first and second trials. (a) Air temperature patterns and (b) relative humidity variations. * represent significant differences (n=7)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3383462/v1/cd55ce852459b5c7cf84aeff.png"},{"id":44605387,"identity":"0e8957f6-8e1b-4118-b12b-9f42016181e6","added_by":"auto","created_at":"2023-10-13 22:20:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":30650,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of (a) light intensity and (b) sunshine hours between the first and second trials. * represent significant differences (n=7)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3383462/v1/f85e28b07e8ef2cc343acaf1.png"},{"id":44605388,"identity":"a4718eb2-c5c2-4873-8e7d-6ea5c25d2c47","added_by":"auto","created_at":"2023-10-13 22:20:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":34078,"visible":true,"origin":"","legend":"\u003cp\u003eVariations in soil fertility under different treatments across three growing periods (n=3)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3383462/v1/6706f26c08aa6affc70e7c6e.png"},{"id":44605389,"identity":"fe403704-9278-464a-a026-c51c087ba488","added_by":"auto","created_at":"2023-10-13 22:20:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":30838,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in below-ground environmental conditions during the second rice growing period, including (a) soil temperature fluctuations and (b) variations in soil electrical conductivity (EC). PNSB: purple non-sulfur bacteria, CR: crop rotation (n=7)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3383462/v1/2fd7491430ad75b08c661f3e.png"},{"id":44605394,"identity":"1a3381e9-276e-4358-aa57-0ae15b1ec3f0","added_by":"auto","created_at":"2023-10-13 22:20:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":19223,"visible":true,"origin":"","legend":"\u003cp\u003eVariations in 5-ALA concentration between PNSB inoculated and uninoculated plants. Means followed by the same letter(s) are not significantly different (p ≤ 0.05) based on Duncan's multiple range test. PNSB: purple non-sulfur bacteria (n=4)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3383462/v1/fd80895131f8a81df479502b.png"},{"id":44608046,"identity":"107047da-1c99-45aa-b336-3dc78d3b2a43","added_by":"auto","created_at":"2023-10-13 22:52:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":28378,"visible":true,"origin":"","legend":"\u003cp\u003eThe changes in plant height until the reproductive stage in (a) the first trial and (b) in the second trial under different treatments. CT: control, PNSB: purple non-sulfur bacteria, CR: crop rotation, and * represent significant differences. (n=10)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3383462/v1/d72b37b2c6849f2f0778bde2.png"},{"id":44607733,"identity":"0ca0a5ad-3d50-41c8-872b-f49ae9276202","added_by":"auto","created_at":"2023-10-13 22:36:48","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":27042,"visible":true,"origin":"","legend":"\u003cp\u003eVariation in leaf chlorophyll content observed until the reproductive stage in (a) the first trial and (b) in the second trial under different treatments. CT: control, PNSB: purple non-sulfur bacteria, CR: crop rotation, and * represent significant differences.(n=10)\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3383462/v1/44eba3dbfa3211bebdf5136c.png"},{"id":44606289,"identity":"a34e321e-488f-4b5d-8544-ef6d1f921454","added_by":"auto","created_at":"2023-10-13 22:28:48","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":21585,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of tiller number until the reproductive stage in (a) the first trial and (b) the second trial under the different treatments. CT: control, PNSB: purple non-sulfur bacteria, CR: crop rotation, and * represent significant differences. (n=10)\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-3383462/v1/a95816156b3092ed89684208.png"},{"id":44605392,"identity":"bec54400-ca26-4ce6-92d3-5d05e7c4daa3","added_by":"auto","created_at":"2023-10-13 22:20:48","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":12009,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in the lodging resistance of rice crop plants under various treatments. Means followed by the same letter(s) are not significantly different (p ≤ 0.05) based on Duncan's multiple range test. CT: control, PNSB: purple non-sulfur bacteria, CR: crop rotation. (n=10)\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-3383462/v1/a68e47c5c5f3fb119307d482.png"},{"id":44605398,"identity":"ea527b3a-748b-408a-91cf-864ef0156e11","added_by":"auto","created_at":"2023-10-13 22:20:48","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":332926,"visible":true,"origin":"","legend":"\u003cp\u003eSynergistic effects of crop rotation and PNSB inoculation on soil nutrients and rice crop productivity. PNSB: purple non-sulfur bacteria, PGPS: plant growth promoting substances, 5-ALA: 5-aminolevulinic acid, IAA: indole-3-acetic acid, NUE: nutrient use efficiency\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-3383462/v1/09237f4fb37121705c50946e.png"},{"id":46228922,"identity":"6777eebc-ff0d-4668-843f-68a4a7480d3b","added_by":"auto","created_at":"2023-11-10 14:34:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2816970,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3383462/v1/c25a81bf-d44a-4d51-a8c7-09c0a558ceb4.pdf"}],"financialInterests":"","formattedTitle":"Unraveling the Novel Synergistic Effects of Crop Rotation and Rhodopseudomonas palustris Inoculation on Rice Productivity and Soil Nutrient Dynamics","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) crop is a vital staple that plays a crucial role in providing food and income for millions of people worldwide (Nguyen and Ferrero \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Khush \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Prasad et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, its sustainability and productivity face increasing threats from various challenges (Kumar et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For instance, the excessive application of chemical fertilizers and pesticides to meet the high demand for rice has resulted in numerous problems, including escalated production costs, environmental deterioration, and adverse impacts on human health (Prashar and Shah \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sharma and Singhvi \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Baweja et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Srivastav \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, the conventional monoculture practice of rice cultivation has also given rise to challenges such as soil quality and fertility depletion, disease outbreaks, pest infestations, and declining yields (Berg \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; He et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Goulart et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, these issues underscore the necessity for alternative and more sustainable farming practices, such as crop rotation, rice-fish cultivation, integrated pest management methods, and organic farming practices.\u003c/p\u003e \u003cp\u003eCrop rotation, an ancient agricultural practice, has been used for centuries to sustainably improve soil quality and crop yield. It refers to the systematic approach of cultivating different crops on the same agricultural land in a planned sequence. Studies have shown that rotating crops helps to enhance soil fertility, regulate pests and diseases, and increase yield (Karlen et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Dadhich et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kakraliya et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). The effectiveness and advantages of crop rotation have been widely acknowledged. For example, in rice-based cropping systems, crop rotation helps to improve soil quality, reduce pests and diseases, and enhance yield (Huang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Adelana et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdditionally, the photosynthetic bacterium \u003cem\u003eRhodopseudomonas palustris\u003c/em\u003e species has been observed to fix nitrogen while producing compounds aiding plant growth, such as 5-aminolevulinic acid (5-ALA) (Kantha et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kantachote et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It has been demonstrated that the use of \u003cem\u003eR. palustris\u003c/em\u003e can increase the growth and yield of a variety of crops, including pak choi (Wong et al. \u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; ShuHua et al. 2015; Xu et al. \u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), stevia (Xu et al. \u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), tobacco (JianFeng et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Su et al. \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), mushroom (Han \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), Chinese dwarf cherry (Yin et al. \u003cspan citationid=\"CR130\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), bean (Batool and Rehman \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and rice (Harada et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Kantha et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kantachote et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Nookongbut et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yen et al. \u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Khuong et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Iwai et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, \u003cem\u003eR. palustris\u003c/em\u003e can act as a biofertilizer, reducing the need for chemical fertilizers while boosting soil health, crop yield, and nutrient assimilation efficiency (Kantha et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kantachote et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNumerous studies have looked at the individual effects of crop rotation (Tanveer et al. \u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Costa et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR132\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)d \u003cem\u003epalustris\u003c/em\u003e inoculation (Wong et al. \u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Hsu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yen et al. \u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) on rice growth and yield; however, their combined effects have not been thoroughly examined. The combined effects of \u003cem\u003eR. palustris\u003c/em\u003e inoculation and crop rotation on rice yield and growth may have significant implications for developing environmentally friendly and economically viable crop management techniques.\u003c/p\u003e \u003cp\u003eTherefore, the current study aims to assess the combined impact of crop rotation and \u003cem\u003eR. palustris\u003c/em\u003e inoculation on the growth and yield of rice crops in field conditions. Additionally, the study will investigate how \u003cem\u003eR. palustris\u003c/em\u003e inoculation and crop rotation affect antioxidant enzyme activity and 5-ALA levels, which are crucial markers of plant growth and stress. In particular, this research will deepen our understanding of how crop rotation and \u003cem\u003eR. palustris\u003c/em\u003e inoculation interact to affect rice productivity and growth and offer suggestions for establishing environmentally sound and long-lasting rice farming practices.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design and setup\u003c/h2\u003e \u003cp\u003eThe current research was conducted at the Practice Farm of the Department of Plant Industry, National Pingtung University of Science and Technology (NPUST), Taiwan, R.O.C. The farm is situated in an open area at coordinates 22\u0026deg;38'54.0\" N and 120\u0026deg;37'01.9\" E. Two fields, each measuring approximately 21 m in length and 9 m in width, were selected for the study area. The blocks were established parallel to each other in the same area under comparable environmental conditions. During the first year of the study, which took place between January and May 2022 (the primary rice-growing season in Taiwan), two fields were utilized to cultivate rice crop. One field was designated as the control group, while the other served as the treatment group. Following rice cultivation, djulis was cultivated in two separate fields from September to December (the primary season for djulis cultivation) of the same year. After djulis were harvested, the stems were crushed into smaller pieces and spread in the soil before final land preparation, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Additionally, in order to enhance soil fertility, any remaining plant materials, including roots, were thoroughly incorporated into the soil through rotovating, ensuring a more balanced nutrient composition and promoting favorable soil conditions for optimal rice growth. In the second year of the study, rice was once again cultivated from January to May 2023. All management practices, including land preparation, planting, and harvesting, were performed uniformly in each block to prevent biases in the results.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Kaohsiung 147 rice crop was transplanted at the 5-leaf stage using the rice transplanter in each field. Ten plants were randomly tagged for field data collection four weeks after transplanting (WAT), while 4 random plants were selected to analyze antioxidant enzyme activities and 5-ALA concentration. The weather conditions, such as air temperature, relative humidity, and light intensity, were monitored using the fully automated KLIMALOG Microclimate Environment Monitoring System provided by Taiwan Hibot Co., Ltd., Kaohsiung, Taiwan R.O.C. Additionally, during the second trial, we extensively monitored the soil environment, specifically focusing on soil temperature and soil electrical conductivity (EC). These parameters were carefully measured using AgriWeather Field Sensor (Beehive Data Technology Co., Ltd., Taipei, Taiwan R.O.C.) to gain insights into the below-ground conditions and their potential impact on rice growth. The data were obtained weekly from the online system and recorded to make informed decisions on management practices. In addition, the soil nutrient analysis was conducted to understand the changes in the soil nutrients under different treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and application of PNSB\u003c/h2\u003e \u003cp\u003eThe biofertilizer containing the \u003cem\u003eR. palustris\u003c/em\u003e species of PNSB was prepared using the initial stock obtained from the Food Industry Research and Development Institute (FIRDI), Taiwan R.O.C (research number PSB32). The culture medium was formulated based on the method described by Lee et al. (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) with some modifications as per the available materials and the suggestions provided by FIRDI. The bacteria were cultivated in a 20 L transparent water bottle and placed in the greenhouse under indirect sunlight for two weeks to promote optimal growth, indicated by the development of a dark maroon color, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The culture bottle was inspected and agitated daily to ensure uniform dispersion of the culture medium for consistent bacterial growth.\u003c/p\u003e \u003cp\u003eAfter 14 days of culture, 10 mL of the stock solution was sampled for laboratory analysis to determine the colony-forming unit (CFU). The original CFU count was determined by the standard plate count (SPC) technique and was adjusted to 2.46 x 10\u003csup\u003e8\u003c/sup\u003e to suit the experimental requirements. The CFU adjustment was implemented to ensure sufficient PNSB in the inoculum, capable of significantly impacting plant growth and yield. This decision was based on previous studies demonstrating the efficacy of similar concentrations of the same bacterial species in promoting plant growth and yield improvement (Yoshida et al. \u003cspan citationid=\"CR131\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Han \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Harada et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Koh and Song \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Yin et al. \u003cspan citationid=\"CR130\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Fan et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wong et al. \u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Hua et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; ShuHua et al. 2015; Su et al. \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nookongbut et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). At 4 WAT, rice plants were inoculated with PNSB, and the inoculation was repeated every two weeks until the early reproductive (heading) stage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant enzyme activity analysis\u003c/h2\u003e \u003cp\u003eThe antioxidant enzyme activity was analyzed from WAT 4 to WAT 12 weekly. The enzymes analyzed were ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), and superoxide dismutase (SOD). The protein content of the enzyme extract was determined using the method of Bradford (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). Samples collected in the field were immediately placed in dry ice and transported to the laboratory for analysis. In the laboratory, a fresh leaf sample (0.05 g) was ground using liquid nitrogen and then homogenized with sodium phosphate buffer (50 mM; pH 6.8 for APX, CAT, GR, and 50 mM; pH 7.4 for SOD) for further grinding before being placed in an ice bath. The solution was then centrifuged at 12,000x g for 20 min (APX, CAT, GR) and 15,000x g for 30 min (SOD) using a Velocity 14R refrigerated Centrifuge (Dymamica Scientific Ltd., Livingston UK) at 4 ℃, and the supernatant was collected.\u003c/p\u003e \u003cp\u003eThe APX activity was analyzed using the method of Nakano and Asada (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). The absorbance was measured at 290 nm for 1 min using a Double Beam U-2900 Spectrophotometer (Hitachi High-Tech Corporation, Japan). As the concentration of ascorbate (AsA) decreased, the absorbance at 290 nm also reduced, and the extinction coefficient of AsA (2.8 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was used to calculate the APX activity. One unit of APX was defined as the amount of enzyme needed to degrade 1 mole of AsA in 1 min. The CAT activity was analyzed using the method of Kato and Shimizu (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). The reduction in hydrogen peroxide amount was measured at 240 nm, and the extinction coefficient (40 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was used to calculate CAT activity. One unit of CAT was defined as the amount of enzyme needed to degrade 1 mole of hydrogen peroxide in 1 min. The GR activity was analyzed using the method of Foster and Hess (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). One unit of GR was defined as the amount of enzyme needed to decrease the absorbance at 340 nm at 1 min. Finally, the SOD activity was analyzed using the method by Paoletti et al. (\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). One unit of SOD was defined as the amount of enzyme that inhibited the rate of NADH oxidation by 50% in the blank sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of 5-aminolevulinic acid\u003c/h2\u003e \u003cp\u003eThe method by Mauzerall and Granick (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e1956\u003c/span\u003e), with slight modifications, was used to determine the concentration of 5-ALA. Initially, a leaf sample weighing 0.05 g was homogenized with sodium acetate buffer (1M; pH 4.7) using a mortar and pestle in an ice bath. The solution was then centrifuged at 10000x g for 5 min at 4\u0026deg;C using a Velocity 14R Refrigerated Centrifuge (Dymamica Scientific Ltd., Livingston, UK), and the resulting supernatant was collected. To this, a 1 mL aliquot of the supernatant was mixed with 50 \u0026micro;l of acetylacetone and incubated at 100\u0026deg;C for 15 min. The solution was then cooled to room temperature, and 3.5 ml of Ehrlich's reagent was added, followed by a 15 min rest. The absorbance of this solution was measured at a wavelength of 556 nm for 20 min using a Double Beam U-2900 Spectrophotometer. Finally, the concentration of 5-ALA was calculated using a standard curve of the 5-ALA reference standard with concentrations ranging from 0 to 30 \u0026micro;g ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCrop management practices\u003c/h2\u003e \u003cp\u003eThe crop management practices used in this study included fertilizer application, irrigation frequency, weeding, and pest and disease management. For dry field preparation, a basal application of farmyard manure was used. Throughout plant growth, the \"Heiwangte No. 43\" compound fertilizer 15-15-15-3(MgO) 50(O.M.), purchased from Taiwan Fertilizer Co., Ltd., was applied in three splits at the early leaf development stage, mid-tillering stage, and early reproductive stage. This application schedule was based on the recommendations of the Miaoli District Agricultural Research and Extension Station, Miaoli County, Taiwan, R.O.C.\u003c/p\u003e \u003cp\u003eThe wet and dry technique was employed to irrigate the rice crop field. This involved irrigating the field for 24 hours and leaving it to dry for three days. This method ensured that the plant roots had access to enough oxygen to carry out respiration while also reducing weed and algae growth. If weeds were still present in the field, manual weeding methods, such as drowning the weeds in mud, were used. For algae growth, \u003cem\u003eBacillus subtilis\u003c/em\u003e was used in addition to the wet and dry method to control the remaining algae. The dead algae also served as a nitrogen source for rice crop plants.\u003c/p\u003e \u003cp\u003ePest and disease control for the rice crop was achieved using organic pesticides, sprayed fortnightly after PNSB treatment was applied. The organic pesticides used were a mixture of saponin, 50% phosphorous acid, and 50% potassium hydroxide. To control snail populations in the early stages of plant growth, organic tea seed cake pallets (16% saponin), an extract from camellia seeds, were spread across the field.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eField data collection\u003c/h2\u003e \u003cp\u003eFrom WAT 4, field data were collected to evaluate the growth and development of the rice crop. This included plant height measurements, tiller number, leaf chlorophyll content, and plant lodging resistance. Ten random plants were selected and marked for fixed data collection weekly until WAT 12 (early reproductive or heading stage) to ensure accuracy in data collection. Prior to data collection, the field was partly dried to ensure accuracy in measurements of plant height and tiller number. Plant height was measured using a simple measuring tape, while individual tillers were carefully counted. For leaf chlorophyll content, the SPAD-502 chlorophyll meter (Konica Minolta, Inc., Japan) was used to determine the relative amount of chlorophyll in the rice crop leaf. Relative chlorophyll content was analyzed at six points on each of the three selected leaves from each plant. Plant lodging resistance was determined using the YYD-IB Plant Stem Strength Tester (Wenzhou Tripod Instrument Manufacturing Co., Ltd., China). Additionally, yield and yield-related traits were evaluated following crop harvest.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData was collected and recorded in Microsoft Excel\u0026reg; 365 (Microsoft Corporation, Washington, DC, USA). Statistical analysis was performed using International Business Machines SPSS Statistics for Windows, version 26 (International Business Machines Corporation, Armonk, New York, USA), with mean comparison conducted using one-way analysis of variance (ANOVA) and mean separation performed using Duncan's multiple-range test. The results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Graphs and charts were created using Origin 2019b software (Origin Lab Corporation, Northampton, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGrowing environment conditions\u003c/h2\u003e \u003cp\u003eThe environmental conditions were extensively monitored in both trials, encompassing a comprehensive range of factors, such as air temperature, relative humidity, light intensity, and duration of sunshine hours. The results revealed similar air temperature patterns in both trials, with minimal variations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Notably, the second trial exhibited significantly higher temperatures at WAT 1, WAT 4, and WAT 5 compared to the first trial. Conversely, the first trial showed a significantly higher temperature at WAT 8 than the second. Despite these differences, the maximum temperature remained consistent between the trials, around 30.3\u0026deg;C and 30.7\u0026deg;C for the first and second trials, respectively. Similarly, the minimum temperature showed slight variation, with 19.6\u0026deg;C in the first trial and 18.8\u0026deg;C in the second. Rice cultivation thrives within an optimal temperature range of 25\u0026ndash;35\u0026deg;C (Hussain et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The minimum temperature that rice can tolerate is 15\u0026deg;C (Chand \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Deviations from this range, either lower or higher, adversely impact crop growth, physiological processes, and, ultimately, yield. In the current study, the data shows a stable and consistent thermal environment for the experimental conditions, with minor variations at specific time points.\u003c/p\u003e \u003cp\u003eThe relative humidity data showed significant variations between the trials (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), with the first trial exhibiting significantly higher than the second, except for specific time points (WAT 13, WAT 14, WAT 15, WAT 16, WAT 18, and WAT 20). The first trial had a higher maximum relative humidity of 85.6% compared to the second trial, which averaged around 76.3%. The minimum relative humidity also varied, with the first trial recording approximately 63.4%, while the second trial had 54.5%. The comparison of relative humidity between the first and second trials highlights significant variations in humidity levels, indicating the influence of environmental conditions on rice growth and development. However, the ideal relative humidity for rice cultivation typically ranges between 60% and 85% (Rathnayake et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In our study, the relative humidity levels for both trials remained within this optimal range, ensuring suitable conditions for the growth and development of the crop.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, the analysis of light intensity also revealed no significant variations between the first and second trials (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The maximum light intensity was approximately 692 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at WAT 9 in the first trial. In contrast, in the second trial, the maximum light intensity recorded was 688.2 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at WAT 11. On the other hand, the minimum light intensity in the first trial was 306 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at WAT 18 and 332 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at WAT 18. Previous studies have shown that the ambient light intensity for rice cultivation typically ranges between 300\u0026ndash;500 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1,\u003c/sup\u003e with \u0026plusmn;\u0026thinsp;100 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e considered acceptable (Huang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Light intensity above this range can adversely affect rice productivity. Fortunately, the light intensity for both trials in our study fell well within the specified range, ensuring favorable rice crop growth and development conditions.\u003c/p\u003e \u003cp\u003eRegarding sunshine hours, the second trial exhibited significantly higher values from WAT 1 to WAT 11, coinciding with crucial stages of plant growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Both trials reached a maximum of 14 hours of sunshine, particularly during the later stages of the experimental period. However, there was a slight disparity in the minimum sunshine hours, with the first trial averaging approximately 8.6 hours and the second trial averaging around 11 hours. Rice plants display optimal growth when exposed to 12\u0026ndash;14 hours of daily sunlight (Jagdish \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In our study, the sunshine hours were consistently within this range, with slight variations. This consistent exposure to the appropriate amount of sunlight provides favorable conditions for the rice plants to thrive and achieve their full growth potential.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSoil nutrient change\u003c/h2\u003e \u003cp\u003eThe soil nutrient change was measured after each trial, and the results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Interestingly, individual applications of PNSB and crop rotation demonstrated improvements in soil fertility, but when these treatments were combined, the effect was even more pronounced. Soil pH plays a crucial role in nutrient availability for plants, and the pH range of 5-7.5 is considered optimal for rice cultivation, although rice plants can tolerate a wide range of soil pH levels. In this study, the soil pH remained within the suitable range (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Previous research has suggested that crop rotation can lead to an increase in soil pH (Ouda et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, contrary to those findings, the current study observed a significant drop in soil pH with crop rotation, and this decline was more pronounced when combined with PNSB inoculation. This discrepancy could be attributed to the unique crop species chosen for rotation in this study, leading to different effects on soil pH compared to previous research (Ouda et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, although not statistically significant, crop rotation positively impacted the soil organic matter (OM) in both fields, which declined in the control field after rice cultivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Conversely, in the PNSB-inoculated field, the bacteria contributed to maintaining soil OM levels. A similar trend was observed for soil P levels, where crop rotation initially increased P concentration in both fields, but later, it declined in both (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). However, the rate of decline in the PNSB-inoculated field was higher, possibly due to PNSB's role in making soil P more available for plant uptake by solubilizing it (Khuong et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Huu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, crop rotation led to enhanced soil K and Ca levels in both fields, but in the PNSB-treated field, these nutrients experienced further enhancement, while there was a decline in the control field, indicating the synergistic effects of crop rotation and PNSB inoculation.\u003c/p\u003e \u003cp\u003eFurthermore, in the control field, with crop rotation, the Mg, Fe, Mn, and Zn concentrations either remained unchanged or decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In contrast, the PNSB inoculated field showed a slight decline in Mn and Zn concentration, with Mg and Fe levels remaining relatively constant. However, the combined treatment resulted in a significantly higher increase in these nutrients compared to the control field, which exhibited only a slight increase. Notably, Cu concentration increased with crop rotation in both fields, with a significant increase in the control field and a significant decline in the PNSB field. Meanwhile, Na concentration declined in both fields after crop rotation but recovered with rice cultivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Overall, the findings highlight the highly effective synergetic effects of crop rotation and PNSB inoculation in enhancing soil nutrient levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant enzyme activity\u003c/h2\u003e \u003cp\u003eThe study analyzed the activity of several antioxidant enzymes, including SOD, APX, CAT, and GR, to determine whether the plants were exposed to stress. The results indicated that PNSB inoculation positively impacted SOD, APX, and GR activity, with decreases of 5%, 3%, and 13%, respectively, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. However, there was an increase in CAT activity in the control field compared to the PNSB-inoculated field. Despite some fluctuations in antioxidant enzyme activity during the experiment, the changes observed were not significant, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These results indicate that the plants were not subjected to any forms of stress that could negatively impact their growth performance and yield. The application of PNSB was found to be beneficial and well-received by the crop.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntioxidant enzyme activity of rice plants under field conditions with and without PNSB treatment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSuperoxide dismutase (SOD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAscorbate peroxidase (APX)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCatalase \u003c/p\u003e \u003cp\u003e(CAT)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGlutathione reductase (GR)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eunits mg\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;1\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eprotein\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\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.139\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.092\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.015\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.016\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePNSB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.132\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.089\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.018\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.014\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (n\u0026thinsp;=\u0026thinsp;5). Means in the same column, followed by the same letter(s), are not significantly different (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) based on Duncan's multiple range test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBelow-ground environment conditions\u003c/h2\u003e \u003cp\u003eIn the second rice cultivation, below-ground environmental conditions were assessed as indicators of plant stress, focusing on soil temperature and soil EC. The results revealed no significant differences in soil temperature between the control and treatment fields, except at WAT 10, WAT 11, WAT 12, and WAT 18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), with maximum temperatures of 26.6\u0026deg;C and 26.3\u0026deg;C in the control and treatment fields, respectively. Similarly, the minimum temperatures were 19.3\u0026deg;C and 19.5\u0026deg;C for the control and treatment fields, respectively, indicating that the temperatures in both fields were maintained within the optimal range of 19.0\u0026deg;C to 27.0\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConversely, soil EC was significantly higher in the treatment field compared to the control field (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), with maximum EC values of 0.43 dS/m and 0.57 dS/m in the control and treatment fields, respectively. The minimum EC values were 0.13 dS/m and 0.25 dS/m in the control and treatment fields, respectively. Studies have shown that the optimum temperature of 25\u0026deg;C for root growth (Arai-Sanoh et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and EC levels below 4 dS/m (Ding et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) is suitable for the healthy growth of rice with a higher yield. Although there were slight variations, the soil temperature and EC levels were within this specified range in the current study (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of 5-aminolevulinic acid\u003c/h2\u003e \u003cp\u003eThe application of PNSB had notable effects on the 5-ALA content in rice crop plants at different time points, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. At WAT 4, a slight difference in 5-ALA concentration was observed between the control and PNSB-treated groups, which was not significant. However, at WAT 9, a significant 12% increase in 5-ALA content was evident in the PNSB-treated plants compared to the control group. This trend continued at WAT 14, with the PNSB-treated group showing a significant 10% highest 5-ALA concentration. These results suggest that PNSB application may positively influence the synthesis or accumulation of 5-ALA in rice crop plants, particularly at later stages of growth as the bacterial population increases. The 5-ALA is a common precursor of tetrapyrroles and is vital as a growth regulator in higher plants (Senge et al. \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Studies have demonstrated the effectiveness of 5-ALA in enhancing photosynthesis and mitigating the detrimental impacts of diverse abiotic stresses on higher plants. In our present study, the application of PNSB resulted in an increase in 5-ALA in plants. This elevation of 5-ALA strengthened the plants' resilience to abiotic stress and substantially improved their overall growth and yield.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAbove-ground plant performance\u003c/h2\u003e \u003cp\u003ePlant height is a crucial agronomic characteristic that significantly impacts rice yield potential (Zhang et al. \u003cspan citationid=\"CR136\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). While the dwarf phenotype offers advantages in preventing lodging, excessively short plants can result in inadequate growth, ultimately hampering the overall yield potential of rice (Zhang et al. \u003cspan citationid=\"CR136\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Striking the right balance in plant height is essential for optimizing rice production and maximizing crop productivity. According to the Kaohsiung District Agricultural Research and Extension Services, the average plant height of the Kaohsiung 147 rice variety is around 98.1 cm in the main growing season. The average plant height for control was 95.4 cm; however, the results of this study show that PNSB inoculation led to a non-significant 4% increase in plant height (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). When PNSB inoculation was combined with crop rotation, there was a significant 9% increase in plant height during the second year compared to crop rotation alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). The notable increase in plant height with PNSB treatment is likely attributed to the enhanced availability of soil nutrients (Ye et al. \u003cspan citationid=\"CR128\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shankar et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) facilitated by the synergistic effects of crop rotation and PNSB inoculation, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. This study presents compelling evidence that the simultaneous implementation of crop rotation and PNSB inoculation yielded positive effects on the growth performance of rice plants.\u003c/p\u003e \u003cp\u003eAdditionally, the one-way ANOVA revealed that the overall plant height in the first year was significantly higher than in the second year, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. These plant height variations could be attributed to differences in sunshine duration. Reduced sunshine hours may result in increased plant height, whereas longer exposure to sunlight might keep plants shorter, as observed in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Therefore, the right balance in the availability of nutrients in the soil and to plants with the right growth environment allows plants to focus more on grain yield rather than vegetative growth, which was evident in this study. Striking this balance proved critical, allowing the plants to channel their energy and resources more efficiently toward producing higher grain yields.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, the study demonstrated that treating rice crops with PNSB and crop rotation significantly affected the leaf chlorophyll level, which is important for plant photosynthesis. The application of PNSB resulted in a significant 4% increase in leaf chlorophyll levels compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). On the other hand, the combined treatment of PNSB and crop rotation led to a significant 3% increase in leaf chlorophyll levels compared to crop rotation alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). Crop rotation alone and the combined treatment of crop rotation and PNSB significantly increased the leaf chlorophyll level by 9% compared to the control and PNSB inoculation, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The one-way ANOVA results suggest that although PNSB inoculation alone can increase leaf chlorophyll levels; however, combining it with crop rotation as treatment can lead to a significantly greater increase in chlorophyll levels. The study unequivocally demonstrates the substantial advantage of crop rotation on leaf chlorophyll concentration. Remarkably, when combined with PNSB inoculation, a noteworthy additional increase in leaf chlorophyll levels is observed. This synergistic effect can be attributed to the improved nutrient availability in the soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), which plays a pivotal role in enhancing plant growth. Adequate nutrient supply boosts leaf chlorophyll levels, a crucial component for efficient photosynthesis. These findings underscore the significance of nutrient management strategies, particularly in the context of combined agricultural practices, and shed light on the mechanisms behind the observed enhancements in plant photosynthetic capacity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, the tiller number in rice crop is also one of the key indicators of grain yield (Li et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR137\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the current study, both PNSB inoculation and crop rotation positively affected the number of tillers in rice plants. Specifically, PNSB inoculation resulted in a 13% increase in the tiller number compared to the control group, although it was not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea). On the other hand, crop rotation led to a significant 107% increase in the tiller number compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). When PNSB inoculation was combined with crop rotation, a significant 27% increase in tiller number was observed compared to crop rotation alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb), and a significant 133% increase compared to PNSB inoculation alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The one-way ANOVA suggests that PNSB inoculation and crop rotation are effective strategies for enhancing the tiller number of rice plants. In particular, crop rotation can substantially increase tiller number, while PNSB inoculation can provide a significant but relatively smaller increase. However, combining PNSB inoculation and crop rotation may offer the most favorable outcomes for enhancing the tiller number in rice cultivation. The significant increase in tiller number could be a result of the enhanced soil nutrient (Tian et al. \u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zha et al. \u003cspan citationid=\"CR134\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR138\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shankar et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) by the combined effects of crop rotation and PNSB inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The observed increase in tiller number directly correlates with higher grain yield (Koprna et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yen et al. \u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), assuming no interference from other biotic or abiotic factors that might impact the crop growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, crop lodging is one of the limiting factors in rice production, and enhancing the lodging resistance of plants can ensure higher grain yield (Zhang et al. \u003cspan citationid=\"CR135\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Shah et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Luo et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tsugawa et al. \u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, both PNSB inoculation and crop rotation significantly increased the lodging resistance of rice plants. The PNSB inoculation resulted in a remarkable 44% increase in lodging resistance compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e11\u003c/span\u003e). In comparison, the crop rotation led to a significant 27% increase compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e11\u003c/span\u003e). Moreover, combining the two treatments resulted in a significant 31% increase in lodging resistance compared to crop rotation alone and a significant 16% increase compared to PNSB inoculation alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe one-way ANOVA analysis shows that both PNSB inoculation and crop rotation can effectively enhance the lodging resistance of rice plants. Though PNSB inoculation may improve lodging resistance more than crop rotation, combining them may offer a greater result in rice cultivation. The observed improvement in lodging resistance in the rice crop can be attributed to the synergistic effects of crop rotation and PNSB inoculation, which led to significant increases in both root growth and tiller numbers. The combination of longer roots, higher tiller numbers, and shorter plants creates favorable conditions to prevent lodging in rice crop plants. This optimal plant structure enhances stability and resilience against bending or collapsing due to wind or heavy rain, thus minimizing yield losses. Consequently, these improvements contribute to increased harvestable yield and overall grain quality. Therefore, this study proves that these combined agricultural practices work harmoniously to enhance the rice plants' overall structural integrity, making them better equipped to withstand lodging and ultimately contributing to more robust and stable crop yields.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBelow-ground plant performance\u003c/h2\u003e \u003cp\u003eThe current study also investigated the effects of PNSB inoculation on the below-ground performance of rice. The results demonstrate that PNSB inoculation significantly enhanced the root growth of rice plants, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Specifically, PNSB inoculation resulted in a 33% increase in root length and a 130% increase in root dry weight compared to the control plants. Although there was a 67% increase in root volume with PNSB inoculation, this increase was not statistically significant. These findings indicate that PNSB inoculation can significantly improve the below-ground performance of rice plants, particularly in terms of root length and dry weight. This could be due to the increase in 5-ALA concentration (An et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), a precursor of tetrapyrroles produced through PNSB inoculation. As a consequence of these enhancements, the rice plants' nutrient uptake capacity is likely to be augmented. With more robust and extensive root systems, plants can access a larger volume of soil, allowing them to absorb essential nutrients more efficiently. This improved nutrient uptake, combined with the growth-promoting effects of PNSB, ultimately leads to a substantial enhancement in the overall growth and productivity of rice crops.\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\u003eRoot growth performance of rice plants inoculated with PNSB compared to uninoculated control in the first trial\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\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRoot length (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoot volume (cm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRoot dry weight (g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePNSB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e333\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (n\u0026thinsp;=\u0026thinsp;3). Means in the same column, followed by the same letter(s), are not significantly different (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) based on Duncan's multiple range test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eYield components and grain metrics\u003c/h2\u003e \u003cp\u003eThe yield-related characteristics of rice, including productive tillers per hill, average grain per hill, grain fertility, and 1000-grain weight, were comprehensively evaluated (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The results unveiled the significant impact of PNSB application on rice crop plants. Specifically, including PNSB led to a significant enhancement of 34% in productive tillers per hill, showcasing its positive influence on tiller formation. Moreover, the implementation of crop rotation resulted in a remarkable 94% increase in productive tillers per hill compared to untreated plants, highlighting the effectiveness of this practice in promoting tiller development. However, when PNSB and crop rotation were combined, the observed increase of 2% in productive tillers per hill was not statistically significant. While the combined effect did not significantly contribute to additional tiller formation compared to crop rotation alone, it is essential to highlight that the synergy between PNSB and crop rotation still yielded superior results compared to PNSB inoculation alone. This integration led to a noteworthy increase of 48% in productive tillers per hill. The outcome underscores the positive impact of the crop rotation and PNSB combination, suggesting a more favorable growing environment and enhanced nutrient supply, likely attributed to the presence of shredded djulis stem.\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\u003eYield characteristics and grain metrics of rice under different treatment conditions\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eYear 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eYear 2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eControl\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePNSB\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCR\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCR\u0026thinsp;+\u0026thinsp;PNSB\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\u003eProductive tillers/ hill (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e98.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage grain/ hill (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e45.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrain fertility (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.57\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e78.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000 grain weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (n\u0026thinsp;=\u0026thinsp;8). Means in the same row, followed by the same letter(s), are not significantly different (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) based on Duncan's multiple range test.\u003c/p\u003e \u003cp\u003eThe average grain per hill analysis revealed substantial effects of the various treatments (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The inoculation of PNSB significantly increased the average grain per hill by 53%, highlighting its positive influence on grain production. Similarly, crop rotation alone resulted in a significant 83% increase in average grain per hill, emphasizing the efficacy of this practice in enhancing grain yield. When PNSB was combined with crop rotation, there was a 13% increase in average grain per hill compared to crop rotation alone. Although this increase was not statistically significant, combining both approaches suggests a potential synergistic effect. Importantly, when comparing the combination of PNSB with crop rotation to PNSB inoculation alone, a significant 34% increase in average grain per hill was observed, further highlighting the added benefit of combining these treatments.\u003c/p\u003e \u003cp\u003eThe analysis of grain fertility revealed significant effects of the different treatments (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). According to Kaohsiung District Agricultural Research and Extension Station (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the grain fertility of the Kaohsiung 147 rice variety is around 68.2% in the main season. However, in this study, grain fertility was around 70.1% in the control field, which was 3% higher than recommended. Conversely, the inoculation of PNSB led to a notable 13% improvement in grain fertility, highlighting its positive impact on this important yield attribute. Additionally, crop rotation alone resulted in a substantial 38% increase in grain fertility, emphasizing its effectiveness in enhancing the reproductive capacity of rice plants.\u003c/p\u003e \u003cp\u003eInterestingly, when comparing crop rotation alone to the combination of crop rotation with PNSB inoculation, a slight variation of approximately 0.30% was observed in grain fertility. Although not statistically significant, this observation suggests a potential synergistic effect between the two treatments, indicating that they may complement each other in enhancing grain fertility. Furthermore, when PNSB was combined with crop rotation, a significant 22% increase in grain fertility was observed compared to PNSB inoculation alone. This finding highlights the positive interaction between PNSB and crop rotation, indicating that their combined application can lead to even greater improvements in grain fertility.\u003c/p\u003e \u003cp\u003eMoreover, the analysis of 1000-grain weight yielded intriguing results (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). According to Kaohsiung District Agricultural Research and Extension Station (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the average 1000-grain weight of the Kaohsiung 147 rice variety is around 23.6 g in the primary growing season. However, in our study, the 1000-grain weight in the control field was around 23.9 g, which was 1% higher than the recommended. Conversely, the inoculation of PNSB led to a modest 2% increase in grain weight compared to the control, but this difference was not statistically significant. In contrast, crop rotation alone showed a significant 13% decrease in grain weight. Likewise, when PNSB inoculation was combined with crop rotation, a significant 12% decrease in grain weight was observed compared to PNSB inoculation alone. Interestingly, comparing the combination of PNSB inoculation with crop rotation to crop rotation alone showed a slight 3% increase in grain weight, although not statistically significant. This result indicates that pursuing higher grain yield may come at the expense of a lower 1000-grain weight. However, there is potential for improvement by implementing additional soil nutrient enhancements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGrain yield and resource allocation\u003c/h2\u003e \u003cp\u003eThe evaluation of the final yield encompassed key parameters such as grain yield, shoot dry weight, and harvest index (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). According to Kaohsiung District Agricultural Research and Extension Station (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the grain yield of Kaohsiung 147 rice variety in the main season is around 6.18 tonnes per hectare. However, our study revealed that the grain yield in the control field was approximately 5.54 tonnes per hectare, which was 12% lower than the recommended yield. This discrepancy could be attributed to long-term monocropping practices, resulting in reduced soil fertility and a less favorable growing environment. Nonetheless, the inoculation of PNSB demonstrated a substantial impact, leading to a 61% significant increase in grain yield.\u003c/p\u003e \u003cp\u003eSimilarly, crop rotation showed a 71% significant increase in grain yield. Remarkably, when PNSB was combined with crop rotation, a significant 19% increase in grain yield was observed compared to crop rotation alone. Furthermore, the combined effects of PNSB and crop rotation resulted in a 27% significant increase in grain yield compared to PNSB inoculation alone. The results highlight that implementing crop rotation or PNSB inoculation individually can lead to higher grain yields in rice, likely attributed to improved soil fertility. Nevertheless, the synergy achieved by combining and applying both treatments demonstrates an even more substantial increase in grain yield, showcasing the potential for enhanced agricultural productivity.\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\u003eGrain yield and resource allocation in rice crop plants under different treatment conditions\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eYear 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eYear 2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eControl\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePNSB\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCR\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCR\u0026thinsp;+\u0026thinsp;PNSB\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\u003eGrain yield (t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShoot dry weight (t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHarvest index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (n\u0026thinsp;=\u0026thinsp;8). Means in the same row, followed by the same letter(s), are not significantly different (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) based on Duncan's multiple range test.\u003c/p\u003e \u003cp\u003eMoreover, the shoot dry weight with PNSB inoculation contributed to a notable 9% increase, even though it was not statistically significant (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similarly, crop rotation exhibited a modest 2% increase in shoot dry weight. However, when PNSB was combined with crop rotation, a significant 26% increase in shoot dry weight was observed compared to crop rotation alone. Furthermore, the combined effects of PNSB and crop rotation yielded a 17% significant increase in shoot dry weight compared to PNSB inoculation alone. These results demonstrate that the combined application of PNSB with crop rotation positively influences shoot dry weight, showcasing a synergistic effect that contributes to improved plant growth and productivity.\u003c/p\u003e \u003cp\u003eFinally, PNSB inoculation resulted in a substantial 46% increase in the harvest index (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similarly, crop rotation showed a 62% significant increase in the harvest index. However, when PNSB was combined with crop rotation, a slight 5% decrease in the harvest index was observed compared to crop rotation alone, which was not a significant decrease. Nevertheless, PNSB and crop rotation combined effects demonstrated a significant 5% increase in the harvest index compared to PNSB inoculation alone. These findings underscore the potential of PNSB inoculation and crop rotation as effective strategies for enhancing rice yield and growth parameters, including grain quality.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGrowing environment conditions\u003c/h2\u003e \u003cp\u003eEnvironmental conditions play a crucial role in the growth and development of rice. Maintaining optimal conditions, including appropriate air temperature, relative humidity, light intensity, and sufficient sunshine hours, promotes healthy rice growth and maximizes crop yield. Rice cultivation requires precise air temperature conditions, and the ideal range for successful growth and development is typically between 25 and 30\u0026deg;C (de los Reyes et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Photosynthesis, a vital process for plant growth, exhibits an optimal air temperature range of 14 to 32\u0026deg;C (Parent et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Deviations beyond this range can negatively impact photosynthetic efficiency. In our study, the air temperature in both trials ranged from 19 to 31\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Although it was slightly outside the optimum range for rice cultivation, it fell within the range for optimal photosynthesis.\u003c/p\u003e \u003cp\u003eRelative humidity also holds significant importance in rice cultivation, impacting yield (Yan et al. \u003cspan citationid=\"CR127\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The ideal relative humidity for rice cultivation typically ranges between 60% and 85% (Rathnayake et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In our study, the relative humidity ranged from 54\u0026ndash;85% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) in both trials, falling within the suitable range. The combined influence of temperature and relative humidity plays a significant role in rice cultivation, acting as the predominant controlling factors, given their spatial and temporal variability (Rathnayake et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, light intensity and duration of sunshine hours are closely intertwined and directly influence rice yield and quality (Liu et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Shafiq et al. \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Rice plants exhibit optimal growth when exposed to 12\u0026ndash;14 hours of daily light (Jagdish \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), with ambient light intensity typically ranging between 300\u0026ndash;500 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1,\u003c/sup\u003e with \u0026plusmn;\u0026thinsp;100 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e considered acceptable (Huang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the current study, the light intensity ranged from 306 to 692 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) for both trials, slightly deviating from the ideal range but mostly within acceptable limits. Similarly, the average daily sunshine hours were around 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) in both trials, aligning with the optimum requirements for rice growth.\u003c/p\u003e \u003cp\u003eOn the other hand, in the first trial, we analyzed antioxidant enzyme activity to assess whether the plants experienced any stress during the growth period, potentially influencing the results. Surprisingly, the results revealed no significant differences in antioxidant enzyme activity between the control and treatment fields, indicating that both sets of plants were not subjected to any noticeable forms of stress (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the second trial, a different approach was taken to assess potential plant stress conditions. Instead of analyzing antioxidant enzyme activity, we focused on the below-ground environment by examining soil temperature and EC levels. This allowed us to gain insights into the influence of below-ground factors on rice growth. In addition to above-ground conditions, factors such as soil temperature and EC levels are key determinants of rice growth and development. Previous studies have indicated that soil temperature significantly affects both grain yield and quality, with rice plants showing optimal root development at around 25 \u0026ordm;C (Arai-Sanoh et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In our study, the average soil temperature in both control and treatment fields was approximately 24 \u0026ordm;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), closely approaching the optimal range.\u003c/p\u003e \u003cp\u003eFurthermore, soil EC is of utmost importance for successful rice cultivation. It directly influences nutrient availability, water movement, and overall soil health, thereby impacting the growth and yield of rice plants. Maintaining an appropriate soil EC level is crucial for optimal nutrient uptake, root development, and overall plant vigor (Ding et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For higher yield, it is recommended to cultivate rice, along with most crops, in low to no-saline soils with an EC below 4 dS/m. In our study, the soil EC levels ranged from 0.13 to 0.57 dS/m in both control and treatment fields, with the treatment field showing significantly higher EC levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSoil nutrient change\u003c/h2\u003e \u003cp\u003eThe combination of crop rotation with PNSB demonstrated remarkable effects on soil nutrient enrichment, as evident from the soil nutrient analysis results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Crop rotation has long been recognized for its ability to improve soil nutrients, as supported by various studies (Francis \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Karlen et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Asseng et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Dhaliwal et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zani et al. \u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To achieve this, selecting appropriate crops that can fix N or enhance nutrient availability is vital. Legumes, for instance, play a valuable role in adding N to the soil through fixation and can be used as green manure (Rangarajan \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, djulis, although not an N-fixing crop like legumes, demonstrated its significance by offering an alternative approach. Even though djulis does not fix N, its tissues contain abundant essential nutrients, including K, P, Mg, Ca, Na, Fe, and Zn (Tsai \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). After harvest, the djulis plant material was shredded and spread across the field before rice planting. This practice allowed the nutrients to gradually leach into the soil, at the same time providing a favorable environment for colonizing beneficial bacteria, such as PNSB, which also assists in enhancing soil nutrients.\u003c/p\u003e \u003cp\u003eThe PNSB then utilizes a specific nitrogenase enzyme to convert atmospheric molecular N into NH\u003csub\u003e3\u003c/sub\u003e or NH4\u003csup\u003e+\u003c/sup\u003e, effectively making N accessible for plant uptake (Franche et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Olivares et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The PNSB also has the ability to improve plant nutrition by mobilizing and increasing the availability of nutrients (Lee et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For example, the elite \u003cem\u003eR. palustris\u003c/em\u003e strain TN110 was found to possess three sets of Mo, V, and Fe nitrogenase gene clusters. As a consequence of this genetic makeup, TN110 released notably higher concentrations of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e compared to the other strains tested in the study (Sakpirom et al. \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, PNSB possesses the capability to solubilize P in the soil. In the context of acid sulfate soil, the combination of low pH and high levels of Al, Fe, H\u003csub\u003e2\u003c/sub\u003eS, and organic acids often leads to reduced P availability due to its immobilization with Al\u003csup\u003e3+\u003c/sup\u003e and Fe\u003csup\u003e2+\u003c/sup\u003e. This results in the formation of insoluble compounds like AlPO\u003csub\u003e4\u003c/sub\u003e\u0026bull;H\u003csub\u003e2\u003c/sub\u003eO and FePO\u003csub\u003e4\u003c/sub\u003e\u0026bull;H\u003csub\u003e2\u003c/sub\u003eO (Yadav and Verma \u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Barrow \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nguyen et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Andrino et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Khuong et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, PNSB can counteract this issue by producing organic acids that interact with these soil minerals, liberating bound P in a soluble form (Khuong et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As a result, P becomes readily accessible for absorption by plant roots, leading to enhanced soil quality, improved nutrient uptake, and, ultimately, increased crop yield. This study is very important as it shows the synergistic benefits of combining crop residues and PNSB application, effectively enriching the soil with essential nutrients (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdditionally, Fe is an element for plant growth and development, as it is essential in the synthesis of chlorophyll, which plays a vital role in photosynthesis (Rout and Sahoo \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kathpalia and Bhatla \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tripathi et al. \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rai et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Arif et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the present study, the PNSB-treated field showed higher Fe accumulation than the control, indicating an enhanced Fe availability in the soil. The increased Fe availability observed in the soil could be attributed to the presence of siderophores, which are small organic molecules secreted by bacteria with a strong affinity for Fe\u003csup\u003e3+\u003c/sup\u003e (Albelda-Berenguer et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kramer et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Khasheii et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Siderophores are produced by almost all known bacterial species (Guerinot \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Ratledge and Dover \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Miethke and Marahiel \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), including PNSB (Nookongbut et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Khuong et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003eb\u003c/span\u003e). In situations where Fe is limited or exists in an insoluble form in the soil, PNSB releases siderophores that form stable complexes by chelating or binding with Fe\u003csup\u003e3+\u003c/sup\u003e. Subsequently, these siderophore-Fe complexes are taken up by PNSB cells, releasing the bound Fe inside the bacterial cells. This efficient process allows PNSB to effectively acquire Fe from the environment, making it available for both the bacteria and the surrounding plants.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eSynergetic effects on rice growth and yield\u003c/h2\u003e \u003cp\u003eThe inoculation of PNSB exhibited a favorable response toward plant growth, and even more promising outcomes were observed with crop rotation alone. However, the most remarkable results were achieved when both practices were combined. The PNSB can promote plant growth by improving nutrient acquisition, producing plant growth-promoting substances, inducing immune system responses, and interacting with the resident microbial community (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e12\u003c/span\u003e). In addition to fixing N for plants, PNSB has been reported to improve N use efficiency (NUE) in plants in the presence of N fertilizer (Wong et al. \u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; ShuHua et al. 2015; Hsu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For example, inoculation of rice with \u003cem\u003eR. capsulatus\u003c/em\u003e DSM155, along with N fertilizer, resulted in a significant 20% increase in nitrogen content within the roots (Elbadry and Elbanna \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). However, it is worth noting that the impact of inoculation was comparatively diminished when N fertilizer was present, in contrast to N-deficient conditions. Likewise, PNSB has also been shown to lower the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e content in crop leaves, which could otherwise have an impact when consumed (Wong et al. \u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, PNSB also produces plant growth-promoting substances such as 5-ALA, IAA (indole-3-acetic acid, siderophores, and exopolymeric substances (Nookongbut et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The IAA, an auxin, exerts multiple positive effects on plant growth and development (Vessey \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Tsavkelova et al. \u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Bending et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Kaymak \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wani et al. \u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), including cell division, root initiation, flowering, fruit setting, ripening, senescence, and gravitropism (Talukdar et al. \u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The PNSB has the capability to synthesize IAA using two pathways: indole-3-pyruvate (IPA) and tryptamine (TAM) pathways, both of which utilize tryptophan as a precursor molecule (Spaepen et al. \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Mujahid et al. 2011). On the other hand, 5-ALA produced by PNSB serves as a significant precursor for tetrapyrrole compounds in plants, including essential components like chlorophyll, heme, and vitamin B12 (Kang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Additionally, 5-ALA acts as a typical plant growth regulator (Wu et al. \u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), exerting influence over various aspects of plant growth, development, and overall yield (Wang et al. \u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Prior research has demonstrated the positive effects of applying 5-ALA to plants, such as improved photosynthesis (Wu et al. \u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), mitigation of abiotic stresses (Wu et al. \u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e), and enhanced fruit quality (Wang et al. \u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Certain photosynthetic bacteria, including specific strains of \u003cem\u003eR. palustris\u003c/em\u003e, can synthesize 5-ALA (Sasaki et al. \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). For instance, under NaCl stress conditions, \u003cem\u003eR. palustris\u003c/em\u003e strains have been found to secrete approximately 2.67 \u0026micro;M of 5-ALA, thereby promoting the growth and biomass of rice roots (Nunkaew et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, 5-ALA also plays a significant role in the regulation of chlorophyll in plants. When applied to plants, 5-ALA promotes the availability of protoporphyrin IX, a key precursor for chlorophyll synthesis (Harada et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As a result, the application of 5-ALA leads to an augmentation in chlorophyll concentration, providing several benefits to plants, including enhanced photosynthetic capacity, increased energy production, and an overall improvement in plant growth. This, in turn, can lead to greater biomass accumulation, increased leaf area, and an overall enhancement of plant performance. Priming plants with 5-ALA has also been shown to boost plant resilience against diverse stresses, encompassing drought, salinity, UV-B radiation, and extreme temperatures. This impact is accomplished by finely adjusting enzyme activities, channel proteins, hormones, signaling molecules, small organic molecules, gene expression, and physiological levels. This regulatory mechanism significantly enhances crucial processes such as photosynthesis, osmoregulation, antioxidant capacity, and N assimilation in plants (Tan et al. \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present study reveals a substantial increase in 5-ALA in plants following PNSB inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This rise in 5-ALA could potentially contribute to elevated leaf chlorophyll content (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). However, it is essential to consider that other nutrients like Mg and Fe also play crucial roles in enhancing chlorophyll synthesis in plants (Farhat et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Consequently, the study's findings demonstrate the synergistic effect of PNSB inoculation and crop rotation, leading to the most favorable overall plant growth performance.\u003c/p\u003e \u003cp\u003eFurthermore, the combined effects of crop rotation and PNSB inoculation, which significantly enhanced rice growth, resulted in a remarkable improvement in yield. Specifically, the integration of crop rotation with PNSB led to an impressive 104% increase in grain yield and a substantial 28% increase in shoot dry weight (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). As a result, the harvest index rose to an outstanding 54%, reflecting the successful synergy between these two agricultural practices in maximizing rice productivity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this research highlights the significant potential of utilizing PNSB in conjunction with crop rotation to foster a synergetic effect on agricultural productivity. The combined approach of PNSB inoculation and crop rotation exhibited a remarkable improvement in soil fertility, as evidenced by enhanced nutrient availability and nutrient cycling processes. Additionally, the presence of PNSB led to a substantial increase in the concentration of 5-ALA in plants, indicating a positive impact on photosynthetic activity and energy production. The observed enhancements in growth parameters, including tiller numbers, leaf chlorophyll content, and lodging resistance, clearly demonstrated the beneficial effects of this integrated approach on rice plants. The positive impact on productive tillers per hill, average grain per hill, and grain fertility ultimately resulted in a significant increase in grain yield and shoot dry weight. The improved harvest index indicates an efficient resource allocation within the crop. These findings suggest that combining PNSB inoculation with crop rotation has promising implications for sustainable agriculture, offering an innovative strategy to address food security challenges. The insights gained from this research could stimulate further investigations in the field of agricultural science and foster the adoption of these novel integrated practices to optimize crop productivity and ensure a more resilient agricultural future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe authors would like to extend their sincere appreciation to the Food Industry Research and Development Institute (FIRDI), situated at 331 Shih-Pin Road, Hsinchu, 300 Taiwan (R.O.C.), for generously supplying the photosynthetic bacteria (Research number PSB32) utilized in this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThe authors did not receive support from any organization for the submitted work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u0026nbsp;\u003c/strong\u003eConceptualization: Laurence Shiva Sundar, Kuei-Shan Yen, Yun-Yang Chao; Methodology: Laurence Shiva Sundar, Kuei-Shan Yen, Yun-Yang Chao; Formal analysis and investigation: Laurence Shiva Sundar, Kuei-Shan Yen, Yao-Tsung Chang; Writing\u0026mdash;original draft preparation: Laurence Shiva Sundar; Writing\u0026mdash;review and editing: Yun-Yang Chao; Funding acquisition: Yun-Yang Chao; Resources: Yao-Tsung Chang, Yun-Yang Chao; Supervision: Yun-Yang Chao\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdelana A, Aduramigba-Modupe V, Oke A, et al (2022) Soil quality assessment under different long-term rice-based cropping systems in a tropical dry savanna ecology of northern Nigeria. 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Plant Production Science 25:311\u0026ndash;319. https://doi.org/10.1080/1343943X.2022.2061538\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"5-aminolevulinic acid, agricultural production, agricultural practices, antioxidant enzymes, food security, photosynthetic bacteria, plant and soil mechanisms, sustainable rice production","lastPublishedDoi":"10.21203/rs.3.rs-3383462/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3383462/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and Aim\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe use of beneficial bacteria, such as purple non-sulfur bacteria (PNSB), has shown great potential for improving plant growth and agricultural production. However, the full extent of their benefits and interaction with agricultural practices is yet to be fully understood. The present study aimed to investigate the synergistic effects of PNSB and crop rotation on rice growth and yield in a field setting and to explore the underlying plant and soil mechanisms by which these practices can benefit farming systems.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment was conducted over two rice cropping seasons, with djulis grown between the rice crops as a rotation crop. Data on the growth and yield of rice was collected and statistically analyzed using a one-way analysis of variance (ANOVA) and Duncan's multiple range test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study demonstrates that PNSB treatment significantly increased the concentration of 5-aminolevulinic acid (5-ALA) in plants, indicating enhanced photosynthesis. Moreover, when combined with crop rotation, PNSB remarkably improved soil fertility. These combined benefits resulted in substantial increases in tiller numbers (163%), leaf chlorophyll content (13%), and lodging resistance (66%) compared to the control. The combined treatment also resulted in higher productive tillers per hill (98%), average grain per hill (106%), and grain fertility (37%). This led to increased grain yield (104%), shoot dry weight (28%), and harvest index (54%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study indicates that a combined approach of PNSB inoculation and crop rotation can effectively enhance the growth and yield of rice plants. These findings have significant implications for sustainable rice production and could potentially contribute to addressing global food security challenges. Improving plant growth and yield could help meet the increasing demand for rice in the face of a growing global population.\u003c/p\u003e","manuscriptTitle":"Unraveling the Novel Synergistic Effects of Crop Rotation and Rhodopseudomonas palustris Inoculation on Rice Productivity and Soil Nutrient Dynamics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-13 22:20:43","doi":"10.21203/rs.3.rs-3383462/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fec5c2b0-55d1-4b0f-94f1-3d7ac3eea0f4","owner":[],"postedDate":"October 13th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-11-10T14:26:23+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-13 22:20:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3383462","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3383462","identity":"rs-3383462","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}