Rhizoglomus clarum inoculation enhances drought tolerance and photosynthetic performance of maize in sterile and natural soils

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Arbuscular mycorrhizal fungi (AMF) have emerged as beneficial symbionts enhancing plant resilience to drought by improving water uptake, nutrient acquisition, and photosynthetic performance. This study evaluated the effects of Rhizoglomus clarum inoculation on maize growth, water status, osmotic adjustment, and chlorophyll a fluorescence under well-watered (WW) and water-deficit (WD) conditions in sterile and natural soils. The experiment was conducted in a greenhouse using a randomized complete block design in a 4 × 2 factorial scheme (soil treatment × water regime), with four replicates. Drought significantly reduced leaf area, shoot and root biomass, and water status. However, R. clarum inoculation attenuated these effects, increasing leaf dry mass by up to 45% and stem dry mass by 100% in under WD. Inoculated plants also showed higher photochemical efficiency (Fv/Fm and PI ABS ) under both water regimes. The strongest responses were observed in natural soil, suggesting synergistic interactions between R. clarum and indigenous microbiota. These results demonstrate that R. clarum enhances maize drought tolerance through coordinated morphological, physiological, and photochemical adjustments. This highlights the potential of species-specific AMF inoculation as a sustainable strategy to improve maize performance under water-limited conditions. Photochemical efficiency Rhizoglomus clarum water restriction Zea mays L Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Water deficit is a major abiotic stress limiting crop growth and productivity worldwide, with serious consequences on food security and the global economy (Irfan et al. 2023 ). Several environmental factors, including reduced precipitation, high salinity, elevated temperatures, and low relative humidity, can increase evaporative demand and lead to water scarcity. In response, plants trigger physiological and biochemical responses (e.g., stomatal closure) to mitigate stress and survive. In addition, plants often rely on beneficial interactions with soil microorganisms to improve their stress tolerance. These associations are recognized as effective strategies to enhance tolerance to drought and nutrient limitations (Kobae 2019 ; Chen et al. 2020 ; Gholinezhad et al.2020; Tang et al. 2022 ; Nie et al. 2024 ). Given the complexity of plant physiological responses to drought, biological strategies that enhance stress tolerance through plant–microbe interactions have gained increasing attention. Among these microorganisms, arbuscular mycorrhizal fungi (AMF) form an ancient and widespread symbiosis with plant roots (Nie et al. 2024 ) that plays a key role in drought tolerance. AMF improve water and nutrient uptake (Parniske 2008 ; Kobae 2019 ; Chen et al. 2020 ; Wang et al. 2020 ; Yang et al. 2025 ), regulate hormonal balance, modulate aquaporin expression, improve root hydraulic conductivity, and strengthen antioxidant defense systems (Sharma et al. 2021 ; Tang et al. 2022 ; Abdelaal et al. 2024 ). These combined effects enhance plant resilience to water deficit. In exchange, plants provide photosynthetically derived carbon to the fungal hyphae, which spread into the soil and access water and essential nutrients such as phosphorus, zinc, calcium, and manganese (Gomes Júnior et al. 2018 ; Kobae 2019 ). Beyond improving nutrition, AMF also help to protect plants from various abiotic and biotic stresses (Brundrett and Tedersoo 2018 ; Moreira et al. 2019 ). Maize, a crop highly sensitive to water stress during early development, benefits significantly from mycorrhizal associations. For example, Funneliformis mosseae can delay the negative effects of drought, increase antioxidant enzyme activity, and reduce the accumulation of abscisic acid and reactive oxygen species (ROS). These effects result in improved growth and biomass (Ren et al. 2019 ; Bahraminia et al. 2020 ). However, most studies have focused on only a few AMF species, particularly Funneliformis and Rhizophagus . As AMF species differ in colonization patterns, host specificity, and stress response mechanisms (Silva et al. 2023 ; Kozikova et al. 2024 ), species-specific studies are essential to understand and exploit their full potential in agriculture. This study evaluated how Rhizoglomus clarum inoculation affects maize physiological, biochemical, and photochemical traits under water deficit in sterile and natural soils. We hypothesize that R. clarum improves drought tolerance by enhancing photochemical efficiency, supporting osmotic adjustment, and promoting vegetative growth. Material and methods Study area and plant material The experiment was conducted in a greenhouse at the State University of Maranhão (UEMA), in São Luís, Maranhão, Brazil (2º31’51” S, 44º18’24” W; at 4 m.a.s.l.), from September to November 2023. The region has a tropical climate with a dry season (Aw) according to the Köppen–Geiger classification (Köpen-Geiger 2015). Early-cycle AG1051 (Seminis®) hybrid maize seeds were used. Soil was collected from the 0–20 cm layer in a preserved vegetation area at the Farm School of UEMA and had the following chemical and physical characteristics: pH (CaCl 2 ) = 4.0; H + Al = 41 cmol c dm − 3 ; Al + 3 = 0 cmol c dm − 3 ; Ca + 2 = 1.0 cmol c dm − 3 ; Mg + 2 = 4.0 cmol c dm − 3 ; K + = 4.6 cmol c dm − 3 ; P (Mehlich) = 5.0 mg dm − 3 ; organic matter = 11.0 g dm − 3 ; base saturation = 19%; and texture = 120 g kg − 1 clay,30 g kg − 1 silt, and 850 g kg − 1 sand (sandy loam soil). Fertilization followed the recommendation for pot experiments under controlled conditions (Novais et al. 1991). We applied 100 mg N, 300 mg P, and 150 mg K per kg of soil at planting. Inoculation of arbuscular mycorrhizal fungi (AMF) Rhizoglomus clarum (CNPAB-A05), obtained from the Embrapa Agrobiology germplasm bank (Seropédica, RJ) was used for AMF inoculations. To ensure successful colonization, we applied 1.43 g of inoculum (containing approximately 100 spores) next to the seeds at planting. Experimental design A randomized block design was used, with four soil treatments: (1) sterile soil - control; (2) sterile soil inoculated with R. clarum ; (3) natural soil without inoculation; and (4) natural soil inoculated with R. clarum . Each treatment was subjected to two water regimes: well-watered (WW) and water deficit (WD), resulting in eight treatment combinations with four replicates. Each experimental unit consisted of a 20 L pot. Five seeds were sown per pot and thinning was carried out on day 10, leaving one plant per pot. Soil for the sterile treatment was autoclaved at 120°C and 1.5 atm for 2 hours. The experiment lasted 51 days, ending at the V5-V6 developmental stage. During the experiment, air temperature (°C), relative humidity (%), and photosynthetically active radiation (µmol m − 2 s − 1 ) were recorded every 15 minutesusing a WatchDog micro station (model 1000 Series, Spectrum Technologies, Inc., Illinois, USA) (Fig. 1 ). Based on temperature and relative humidity data, vapor pressure deficit (VPD air ) was calculated according to Jones (1992) using the following formula : \(\:\text{F}\text{o}\text{r}\text{m}\text{u}\text{l}\text{a}\:1.\:{VPD}_{air}=0.61137\times\:\text{exp}\left(\frac{\begin{array}{c}17.502\:\times\:\:\text{T}\end{array}}{240.97+\text{T}}\right)\times\:\:(1-\frac{\text{R}\text{H}}{100}\) ) Where: VPD is the vapor pressure deficit (MPa); exp denotes the exponential function; T is the temperature (°C); and RH is the relative humidity (%). Soil moisture was maintained at 90% of field capacity (FC) through daily irrigation. Soil moisture levels were monitored using an RS485 TH-HMI043 sensor (ComWinTop, Mainland, China), following the methodology of Morales et al. ( 2015 ). Readings were taken daily in all experimental units. Water restriction was imposed at 46 days after planting (DAP), between the V5 and V6 stages. This period is critical, as environmental stress can reduce the potential number of rows per ear (Magalhães and Durães 2006 ). Morpho-anatomical measurements At 51 DAP, leaf area (LA, cm 2 ) was estimated according to the method of Radford ( 1967 ), using the formula: Formula 2. \(\:LA=K\:(LL\:\times\:\:ML)\) Where LA is the leaf area (cm 2 ), K is the constant (0.75), LL is the leaf length (cm), and ML is the maximum leaf width (cm). On the same day, plants were harvested and separated into leaves, stems, and roots. Each plant part was dried in a forced-air circulation oven at 70°C until constant weight. The dry mass of leaves (LDM), stems (SDM), and roots (RDM) was then determined. Water status Relative water content (RWC) was measured at 51 DAP using ten discs from a fully expanded leaf with a visible collar, ligule, and auricle. Fresh mass (FM, g) was determined immediately using an analytical balance. The discs were then rehydrated in deionized water for 24 h in the dark. Afterwards, excess surface water was removed with paper towels and turgid mass (TM, g) was recorded. To determine dry mass (DM, g), the discs were dried in a forced-air circulation oven at 65°C for 48 hours. RWC was calculated following Deng et al. ( 2024 ) using the formula: Formula 3. \(\:RCW\:\left(\%\right)=\frac{FM-TM}{TM-DM}\:\:\times\:100\) Shoot water content (SW, g H 2 O) was estimated as the difference between the total fresh mass (leaves + stems) and total dry mass. Proline and total soluble carbohydrates Proline was determined using 0.05 g of dry leaf tissue from the same samples used in the water status analysis. The extract was prepared with 2 mL of 3% sulfosalicylic acid. Proline content was determined according to the method of Bates et al. ( 1973 ), based on absorbance at 520 nm using a UV-5100 UV-Vis spectrophotometer (Shanghai Metash Instruments Co., Shanghai, China). The proline concentration (µmol g − 1 DM) values were calculated from a standard curve using L-proline. Total soluble carbohydrate (TSC) content was determined using 0.020 g of dry leaf mass, following the method of Dubois et al. ( 1956 ). Absorbance was read at 490 nm using the same spectrophotometer, and concentrations (mg g − 1 DM) were calculated from a standard glucose curve. Green intensity index and chlorophyll a fluorescence Green intensity index (SPAD) was measured using a portable chlorophyll meter (model SPAD-502, Minolta, Japan). Measurements were taken on a fully expanded leaf between 8:00–10:00 a.m. and 12:00–2:00 p.m. during five days of water deficit. Ten readings per leaf were recorded, and the mean value per plant was calculated. Chlorophyll a fluorescence was measured on the same leaves using a portable fluorometer (FluorPen FP 110, Photon Systems Instruments, Drásov, Czech Republic). Readings were taken from 8:00–9:00 a.m. and 12:00–1:00 p.m., after a 30-minute dark adaptation using leaf clips placed away from the midrib. A saturating light pulse (3000 µmol m − 2 s − 1 , 650 nm, 1 second) was then applied to assess fluorescence transients, based on the OJIP test (Strasser et al. 1995). The following parameters were obtained: initial fluorescence (F 0 ), variable fluorescence (Fv), maximum fluorescence (Fm), primary fluorescence (Fv/F 0 ), maximum quantum efficiency of photosystem II (Fv/Fm), and photosynthetic performance index (PI ABS ). Photosynthetic pigments The chlorophyll a , chlorophyll b, and carotenoid contents were quantified from five leaf discs (0.6 cm² each) collected from fresh tissues. The discs were placed in glass tubes wrapped in aluminum foil with 5 mL of 80% acetone and stored in the dark at 4°C for 72 hours. Absorbance of the extracts was measured at 480, 645, and 663 nm using a UV-5100 UV-Vis spectrophotometer (Shanghai Metash Instruments Co., Shanghai, China). Pigment concentrations were calculated using the equations of Lichtenthaler ( 1987 ). Spore extraction and morphological identification AMF glomerospores were extracted from 50 g of air-dried soil collected from the experimental treatments using the wet sieving and decanting method described by Gerdemann and Nicolson ( 1963 ), followed by centrifugation in a sucrose solution according to Jenkins (1962). Spores were quantified on channeled plates under a stereomicroscope at 40× magnification. For taxonomic identification, glomerospores were mounted on microscope slides with polyvinyl lacto-glycerol (PVLG) and a mixture of PVLG with Melzer’s reagent. They were examined under a compound microscope and identified based on morphological characteristics following Schenck and Pérez ( 1990 ) and using specialized online databases (INVAM, http://invam.caf.wvu.edu ). Colonization rate At the end of the water restriction period, maize roots were collected and thoroughly washed to remove adhering soil particles. Root samples were cleared in 5% (w/v) potassium hydroxide (KOH) and stained with trypan blue following the method of Phillips and Hayman ( 1970 ). The stained roots were examined under a light microscope to confirm the presence of arbuscular mycorrhizal colonization. Plants were considered colonized when arbuscules, vesicles, spores, or hyphae were observed. Statistical analysis All data were tested for normality using the Shapiro-Wilk test. Variables meeting normality assumptions were subjected to analysis of variance (ANOVA), and the means were compared using Tukey test at a 5% significance level. Statistical analyses were performed in R software (version 3.4.1; R Core Team, 2023 ) using the “ExpDes.pt” (Ferreira et al. 2018 ) and “factoextra” (Kassambara 2020 ) packages. Results Identification of AMF species and colonization rate Eight AMF species were recovered from the natural soil used in the experiment. Based on morphological characteristics, the following species were identified: Acaulospora morrowiae Spain & N.C. Schenck, Acaulospora scrobiculata Trappe, Acaulospora foveate Stürmer & Morton, Ambispora appendicula Spain, Sieverd. & N.C. Schenck) C. Walker, and four unidentified Glomus morphotypes ( Glomus sp1, Glomus sp2, Glomus sp3 [ glomerulatum like], and Glomus sp4). Root colonization was significantly affected by soil condition and AMF inoculation (p < 0.001). Under water deficit, NAT + RHIZO treatment showed the highest colonization rate (40.6%), representing a 1.92-fold increase compared to STE + RHIZO and a 1.41-fold increase compared to NAT alone (Fig. 2 ). Leaf area and dry biomass A significant difference in leaf area (LA) was observed among treatments (p < 0.03). Under well-watered conditions, the NAT + RHIZO treatment showed the highest mean LA (736.3 cm²). Under water deficit, NAT + RHIZO had a 36.8% increase in LA compared to the control (WD_STE) (Fig. 3A). Figure 3 Growth parameters of maize plants under different soil and water conditions. (A) Leaf area, (B) leaf dry mass, (C) stem dry mass, and (D) root dry mass. WW: well-watered; WD: water deficit; STE + RHIZO: sterile soil inoculated with Rhizoglomus clarum ; NAT: natural soil; NAT + RHIZO: natural soil inoculated with R. clarum . Data are means ± standard error (n = 4). Significant differences (ANOVA, Tukey test, p < 0.05) are shown by letters. Capital letters compare treatments under WW and lower case letters compare treatments under WD. Asterisks indicate a significant difference between water regimes within the same treatment. The columns without letters did not show significance Leaf dry mass (LDM), stem dry mass (SDM), and root dry mass (RDM) differed among treatments (p < 0.00) (Fig. 3B-D). Under water deficit, LDM in the NAT + RHIZO treatment was 45.4% higher than in the control STE. Under well-watered conditions, NAT + RHIZO also showed the highest LDM with 43.7% increase compared to STE (Fig. 3B). For SDM, NAT + RHIZO resulted in 100% increase over the control STE under water deficit and 63.16% increase in well-watered conditions (Fig. 3C). RDM was 43.7% higher in STE + RHIZO when compared to NAT under water deficit (Fig. 3D). Water status Relative water content (RWC) and shoot water (SW) differed significantly between the water regimes (p < 0.00) (Fig. 4 A). Under well-watered conditions, SW was higher in the NAT + RHIZO treatment compared to STE and STE + RHIZO, with icreases of 39.6% and 38.7%, respectively. In contrast, we observed no differences in SW among treatments under water deficit conditions (p > 0.075) (Fig. 4 B). Total soluble carbohydrate, proline, and photosynthetic pigment contents Total soluble carbohydrate (TSC) content differed between the water regimes (p < 0.001) (Fig. 5 A). In the STE + RHIZO treatment, TSC was 114.1% higher under well-watered conditions than under water deficit RHIZO. We observed a similar trend in the NAT + RHIZO treatment, with a 95.3% increase under irrigation RHIZO. Proline content varied among treatments only under water deficit (p < 0.001), with the NAT treatment showing the highest levels (Fig. 5 B). Concerning the photosynthetic pigments, carotenoid content differed significantly only among treatments under well-watered conditions (p 0.05) (Supplementary Table S1 ). Stomatal density Abaxial stomatal density differed among treatments under both well-watered (p < 0.001) and water-deficit (p < 0.03) conditions. Under water deficit, STE had the lowest mean density (Fig. 6 A). For the adaxial surface, significant differences were observed only under well-watered conditions (p 0.01) (Fig. 6 C and D). Chlorophyll a fluorescence The maximum quantum yield of photosystem II (Fv/Fm) differed between water regimes in the morning (p < 0.01). NAT + RHIZO and STE + RHIZO treatments showed the highest Fv/Fm values in well-watered plants, with the former standing out (i.e., 50% higher than the control) (Fig. 7 A). STE + RHIZO also showed a significant increase of 14.7% relative to the control. In the afternoon, significant differences were observed only under water deficit conditions (p < 0.001). In the NAT + RHIZO and WD_STE + RHIZO treatments had Fv/Fm values 52.9% and 56.6% higher, respectively, than STE (Fig. 7 B). Primary fluorescence efficiency (Fv/F o ) differed among treatments only under water-deficit, both in the morning and afternoon (p < 0.01) (Fig. 7 C). In the afternoon, Fv/F o was higher in the STE + RHIZO and NAT + RHIZO treatments, with the latter showing a 1.26-fold increase compared to STE (Fig. 7 D). Performance index (PI ABS ) differed among water-deficit treatments in the morning (p < 0.00) (Fig. 7 E) and well-watered treatments in the afternoon (p < 0.00) (Fig. 7 F). WD_NAT had the highest mean and NAT + RIZHO also differed between water regimes (Fig. 7 E). In the afternoon, WW_NAT + RHIZO had the highest mean (43.9% increase compared to WW_STE) and the only treatment showing differences between water regimes (Fig. 7 F). Green intensity index (SPAD) values were similar among treatments and water regimes (p > 0.05) (Supplementary Table S2). Discussion Drought negatively affects maize growth by reducing water availability, limiting leaf and root development, and decreasing photosynthetic rate and water use efficiency, ultimately impairing plant productivity (Avramova et al. 2016 ). In this context, AMF have emerged as beneficial symbionts that help plants cope with water stress. Here, the successful colonization of maize roots by R. clarum confirmed the establishment of a functional mycorrhizal symbiosis. Although drought reduces the colonization rate (Begum et al. 2019 ), inoculation of R. clarum in natural soil increased the mycorrhizal colonization rate, resulting in similar levels between irrigated and water-stressed treatments. This association led to improvements in key growth and physiological traits. Inoculated plants in natural soil had increased leaf area under both well-watered and water-deficit conditions (Fig. 3A). A larger leaf area contributes to improved light interception and photosynthetic capacity (Luiz et al. 2006 ; Pinheiro et al. 2020 ), which are critical for maintaining growth under drought. Similar increases in leaf area have been observed in other studies involving maize and AMF under stress, reinforcing their positive role in leaf development (Agbodjato et al. 2022 ). Inoculation with R. clarum significantly increased the dry biomass of maize shoots, stems, and roots (Fig. 3B-D). AMF can enhance plant growth by improving water and nutrient uptake through their hyphae, stimulating root growth and mitigating drought (Tian et al. 2011 ; Ren et al. 2019 ). The stronger biomass response in NAT + RHIZO compared to STE + RHIZO suggests a synergistic interaction between the inoculated R. clarum and native microbial communities. Indigenous AMF and beneficial rhizobacteria may facilitate colonization, enhance hyphal proliferation, or contribute complementary drought-mitigation mechanisms, a pattern increasingly recognized in studies on AMF functioning in natural soils (Sharma et al. 2021 ; Abdelaal et al. 2024 ). Similar benefits of AMF have been reported under other abiotic stress conditions, such as salinity in maize (Xu, Lu and Tong 2018 ). Contrary to previous studies reporting higher relative water content in AMF-inoculated plants (Zhu et al. 2011 ; Santos et al. 2023 ), we observed no significant differences under our experimental conditions (Fig. 4 A). Relative water content is an important indicator of plant water status and is closely linked to photosynthesis, biomass accumulation, and productivity (Ashraf et al. 2020 ). In contrast, shoot water content was positively influenced by R. clarum inoculation, particularly under well-watered conditions (Fig. 4 B). Higher shoot water in inoculated plants suggests that AMF may help maintain leaf hydration. Similar results were reported by Ait-El-Mokhtar et al. ( 2020 ), who observed improved leaf water content under drought. This improvement is likely related to increased hydraulic conductivity and more efficient water absorption mediated by AMF. Both proline and total carbohydrate contents were higher in AMF-inoculated plants (Fig. 5 A and B), indicating enhanced osmotic adjustment. Proline helps plants maintain osmotic homeostasis, stabilizes proteins and membranes, and mitigates oxidative damage under water stress (Laxa et al. 2019 ; Sadak et al. 2019 ). Soluble carbohydrates also contribute to stress tolerance by acting as antioxidants and regulating reactive oxygen species (ROS) signaling (Begum et al. 2019 ). The accumulation of these compatible solutes likely contributed to maintaining water status and improving plant resilience under drought, consistent with findings reported by Begum et al. ( 2019 ). Carotenoid levels were higher in plants grown in natural soil (NAT) compared to those in sterile soil STE (Fig. 5 C), suggesting that native communities of arbuscular mycorrhizal fungi, along with the existing soil microbiota to photoprotection. Carotenoids protect plants against oxidative stress by quenching ROS and dissipating excess light energy, thus preventing photoinhibition (Hazrati et al. 2016 ). Our results align with studies showing that native AMF can enhance antioxidant capacity through functional complementarity (Begum et al. 2019 ; Ye et al. 2023 ). Despite evidence that drought can reduce chlorophyll content (Avramova et al. 2016 ) and that AMF may improve green intensity index under stress (Yan et al. 2022 ), no significant differences in chlorophyll contents or SPAD values were observed among treatments in this study. Chlorophyll a fluorescence is widely used to assess the efficiency of photosystem II (PSII) and plant responses to stress (Murchie and Lawson 2013 ; Ye, Wang and Li 2022 ). Drought reduced PSII efficiency as indicated by declines in Fv/Fm, Fv/F o , and performance index (PI ABS ) (Fig. 7 ). The observed Fv/Fm values were below the typical range for healthy plants (0.75–0.85), confirming the severity of stress (Chen et al. 2016 ). However, inoculation with R. clarum reduced the magnitude of this decrease, leading to higher Fv/Fm values ​​compared to non-inoculated plants (Fig. 7 ). These results suggest that AMF symbiosis attenuated photoinhibition and contributed to the partial maintenance of photosynthetic activity, likely by enhancing water uptake, stabilizing membrane integrity, and reducing oxidative stress (Tang et al. 2022 ; Rasouli et al. 2023 ; Huang et al. 2024 ). Similar protective effects have been reported in maize and other crops under drought and heat stress (Zhu et al. 2011 ; Porcel et al. 2015 ; Maia et al. 2020 ; Zhang et al. 2021 ). Taken together, this study demonstrates that R. clarum inoculation enhances maize drought tolerance through coordinated improvements in growth, water status, osmotic adjustment, and photochemical efficiency. The stronger effects observed in natural soil suggest a synergistic interaction between R. clarum and indigenous microbial communities, reinforcing the value of AMF consortia in field conditions. AMF-inoculated plants maintained higher biomass, accumulated more compatible solutes, and showed improved photosystem II performance under stress, supporting the view that AMF act as holistic modulators of plant resilience. These results contribute to a growing of evidence that AMF can mitigate abiotic stress beyond nutrient uptake, with implications for sustainable crop production under water-limited conditions (Tang et al. 2022 ; Abdelaal et al. 2024 ; Yang et al. 2025 ). Although our study did not directly assess molecular mechanisms, previous research suggests that AMF can enhance drought tolerance by increasing root hydraulic conductivity, modulating aquaporin expression, and regulating hormonal pathways such as abscisic acid and jasmonates (Tang et al. 2022 ; Ye et al. 2023 ). These adjustments collectively improve water status and carbon assimilation under limited moisture conditions. Future research integrating transcriptomics and root hydraulic analyses could elucidate the specific pathways by which R. clarum promotes adaptation to drought in natural soils. Conclusion This study underscores the potential of R. clarum as a microbial tool for enhancing maize resilience under drought. Beyond confirming the functional role of AMF, our findings point to the importance of species-specific interactions and soil context in shaping plant responses to water stress. The synergistic effects observed in natural soil highlight the value of local microbial communities to optimize inoculant performance. Integrating AMF-based inoculants into agricultural systems may offer a scalable, low-input, and sustainable approach to improve crop stability under increasingly variable climate conditions. Abbreviations AMF Arbuscular mycorrhizal fungi Chl a Chlorophyll a Chl b Chlorophyll b DAP Days after planting DM Dry mass F₀ Minimum fluorescence yield FC Field capacity FM Fresh mass Fm Maximum fluorescence yield Fv/F₀ Initial photochemical efficiency of PSII Fv/Fm Maximum quantum efficiency of PSII LA Leaf area LDM Leaf dry mass NAT Natural soil NAT + RHIZO Natural soil + Rhizoglomus clarum inoculation PI ABS Performance index based on absorption PSII Photosystem II RDM Root dry mass RHIZO Rhizoglomus clarum ROS Reactive oxygen species RWC Relative water content SDM Stem dry mass SPAD Soil and Plant Analyzer Development Index STE Sterile soil STE + RHIZO Sterile soil + Rhizoglomus clarum inoculation SW Shoot water TM Turgor mass TSC Total soluble carbohydrate VPD air Vapor pressure deficit of air WD Water deficit WW Well–watered. Declarations Conflict of interest The authors declare no competing interests. Funding This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contribution TMF and CPN conceived the study and supervised the project. TMF, NBC, SOMJ, CPN, and MRSAL designed and performed experiments. MRSAL and NBC wrote the manuscript. MRSAL, NBC, PCS, DBC, SOMJ, and JRA assisted in performing the experiments. TRC, FOR, WPR, PHAC, ION, CPN, FAMAF, and TMF review and editing manuscript. MRSAL and NBC Review and editing finally for submission. All authors read and approved the final manuscript. Acknowledgement This study was carried out support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. The authors also acknowledge the support of the National Council for Scientific and Technological Development (CNPq): PQ 312653/2025-5 awarded to Tiago M. Ferraz, PQ 307349/2023-3 awarded to Thais R. Corrêa, PQ 305522/2025-6 awarded to Fabrício de O. 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Probing photosynthesis 25:445–483 Tang H et al (2022) The Critical Role of Arbuscular Mycorrhizal Fungi to Improve Drought Tolerance and Nitrogen Use Efficiency in Crops. Front Plant Sci1 3:919166. https://doi.org/10.3389/fpls.2022.919166 Tian H et al (2011) Spatio-temporal dynamics of an indigenous arbuscular mycorrhizal fungal community in an intensively managed maize agroecosystem in North China. Appl Soil Ecol 47(3):141–152. https://doi.org/10.1016/j.apsoil.2011.01.002 Wang S et al (2020) Functional analysis of the Os NPF4.5 nitrate transporter reveals a conserved mycorrhizal pathway of nitrogen acquisition in plants. PNAS 117(28):16649–16659. https://doi.org/10.1073/pnas.2000926117 Xu H, Lu Y, Tong S (2018) Effects of AMF on photosynthesis and chlorophyll fluorescence of maize under salt stress. Emir J Food Agric 30:199–204 Yan Q et al (2022) Arbuscular mycorrhizal fungi improve the growth and drought tolerance of Cinnamomum migao by enhancing physio-biochemical responses. Ecol Evol 12(7):e9091. https://doi.org/10.1002/ece3.9091 Yang X et al (2025) How do arbuscular mycorrhizal fungi enhance drought resistance of Leymus chinensis? BMC Plant Biol 25:453. https://doi.org/10.1186/s12870-025-06412-1 Ye Q, Wang H, Li H (2022) Arbuscular mycorrhizal fungi improve growth, photosynthetic activity, and chlorophyll fluorescence of vitis vinifera l. cv. ecolly under drought stress. Agronomy 12(7):1563. https://doi.org/10.3390/agronomy12071563 Ye Q, Wang H, Li H (2023) Arbuscular Mycorrhizal Fungi Enhance Drought Stress Tolerance by Regulating Osmotic Balance, the Antioxidant System, and the Expression of Drought-Responsive Genes in Vitis vinifera L . Aust J Grape Wine Res 2023:7208341. https://doi.org/10.1155/2023/7208341 Zhang R et al (2021) Comprehensive utilization of corn starch processing by-products: a review. Grain. Oil Sci Technol 4:89–107. https://doi.org/10.1016/j.gaost.2021.08.003 Zhu X-C et al (2011) arbuscular mycorrhizal fungus on photosynthesis and water status of maize under high temperature stress. Plant soil 346:189–199. https://doi.org/10.1007/s11104-011-0809-8 Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile.docx 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8534257","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594436000,"identity":"d7a4a9f8-310a-4809-9558-fbbec3535a3d","order_by":0,"name":"Maria Rita da Silva Andrade Leonel","email":"","orcid":"","institution":"Universidade Estadual do Maranhão","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Rita da Silva Andrade","lastName":"Leonel","suffix":""},{"id":594436001,"identity":"f8be50c0-6252-4bf6-b580-e0e9f495f29f","order_by":1,"name":"Niedja Bezerra 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Aragão","lastName":"Catunda","suffix":""},{"id":594436012,"identity":"ff017564-ee71-45d5-a15d-64a81bff28be","order_by":12,"name":"Ivaneide de Oliveira Nascimento","email":"","orcid":"","institution":"Universidade Estadual da Região Tocantina do Maranhão","correspondingAuthor":false,"prefix":"","firstName":"Ivaneide","middleName":"de Oliveira","lastName":"Nascimento","suffix":""},{"id":594436013,"identity":"573404fa-6540-4403-a857-df618dc4bac4","order_by":13,"name":"Tiago Massi Ferraz","email":"","orcid":"","institution":"Universidade Estadual do Maranhão","correspondingAuthor":false,"prefix":"","firstName":"Tiago","middleName":"Massi","lastName":"Ferraz","suffix":""}],"badges":[],"createdAt":"2026-01-06 18:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8534257/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8534257/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103396105,"identity":"09dd45c1-cbb4-4512-94c0-2221ac825e27","added_by":"auto","created_at":"2026-02-25 08:46:45","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":359486,"visible":true,"origin":"","legend":"\u003cp\u003eClimate conditions during the experimental period (September to November 2023), including maximum (T max), average (T ave), and minimum (T min) air temperature, and maximum (RH max), average (RH ave), and minimum (RH min) relative humidity. The black arrow indicates the planting date. The white and gray arrows indicate the beginning and end of the water-deficit period, respectively.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8534257/v1/5a32988b3d53ee9bef04de1b.jpeg"},{"id":103506943,"identity":"523a0584-593e-4e60-82b1-412b16e1dec2","added_by":"auto","created_at":"2026-02-26 13:40:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":139160,"visible":true,"origin":"","legend":"\u003cp\u003eRoot colonization rate of maize under different soil and water conditions. WW: well-watered; WD: water deficit; STE+RHIZO: sterile soil inoculated with \u003cem\u003eRhizoglomus clarum\u003c/em\u003e; NAT: natural soil; NAT+RHIZO: natural soil inoculated with \u003cem\u003eR. clarum\u003c/em\u003e. Data are means ± standard error (n = 4). Significant differences (ANOVA, Tukey test, p \u0026lt; 0.05) are shown by letters. Capital letters compare treatments under WW and lower case letters compare treatments under WD. The asterisk indicates a significant difference between water regimes within the same treatment.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8534257/v1/4a3d2b8d3d32dcd87db27b21.png"},{"id":103396106,"identity":"8e64910c-58cb-4cf2-9102-d24e689642a9","added_by":"auto","created_at":"2026-02-25 08:46:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":319308,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth parameters of maize plants under different soil and water conditions. (A) Leaf area, (B) leaf dry mass, (C) stem dry mass, and (D) root dry mass. WW: well-watered; WD: water deficit; STE+RHIZO: sterile soil inoculated with \u003cem\u003eRhizoglomus clarum\u003c/em\u003e; NAT: natural soil; NAT+RHIZO: natural soil inoculated with \u003cem\u003eR. clarum\u003c/em\u003e. Data are means ± standard error (n = 4). Significant differences (ANOVA, Tukey test, p \u0026lt; 0.05) are shown by letters. Capital letters compare treatments under WW and lower case letters compare treatments under WD. Asterisks indicate a significant difference between water regimes within the same treatment. The columns without letters did not show significance\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8534257/v1/f6a9a78c9722cb97f19a2984.png"},{"id":103507638,"identity":"a2eb3e28-927d-4c53-85cc-88919bc83405","added_by":"auto","created_at":"2026-02-26 13:42:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":190312,"visible":true,"origin":"","legend":"\u003cp\u003eWater status indicators of\u003cstrong\u003e \u003c/strong\u003emaize plants under different soil and water conditions. (A) Relative water content (RWC) and \u0026nbsp;(B) shoot water (SW). WW: well-watered; WD: water deficit; STE+RHIZO: sterile soil inoculated with \u003cem\u003eRhizoglomus clarum\u003c/em\u003e; NAT: natural soil; NAT+RHIZO: natural soil inoculated with \u003cem\u003eR. clarum.\u003c/em\u003e Data are means ± standard error (n = 4). Representation of the simple effect in the comparison of treatments, significant differences (Tukey test, p \u0026lt; 0.05) are indicated by different letters\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8534257/v1/7fc41199e16f404143857dff.png"},{"id":103396109,"identity":"b688a0e4-c42c-4bee-9a9f-f8eafcd346f7","added_by":"auto","created_at":"2026-02-25 08:46:46","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":293149,"visible":true,"origin":"","legend":"\u003cp\u003eBiochemical stress indicators in maize plants under different soil and water conditions. (A) Total soluble carbohydrate, (B) proline, and (C) carotenoid contents. WW: well-watered; WD: water deficit; STE+RHIZO: sterile soil inoculated with \u003cem\u003eRhizoglomus clarum\u003c/em\u003e; NAT: natural soil; NAT+RHIZO: natural soil inoculated with \u003cem\u003eR. clarum\u003c/em\u003e. Data are means ± standard error (n = 4). Representation of the simple effect in the comparison of treatments, significant differences (Tukey test, p \u0026lt; 0.05) are indicated by different letters\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8534257/v1/191d611257cceadbd06b78ce.jpeg"},{"id":103396103,"identity":"58b4d8dc-5648-408d-b79e-cd9099efcfed","added_by":"auto","created_at":"2026-02-25 08:46:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":410571,"visible":true,"origin":"","legend":"\u003cp\u003eStomatal density and functionality on the leaf surfaces of maize under different soil and water conditions. (A) Abaxial and (B) adaxial stomatal density, and (C) abaxial and (D) adaxial stomatal functionality. WW: well-watered; WD: water deficit; STE+RHIZO: sterile soil inoculated with \u003cem\u003eRhizoglomus clarum\u003c/em\u003e; NAT: natural soil; NAT+RHIZO: natural soil inoculated with \u003cem\u003eR. clarum\u003c/em\u003e. Data are means ± standard error (n = 4). Significant differences (Tukey test, p \u0026lt; 0.05) are shown by letters. Capital letters compare treatments under WW and lower case letters compare treatments under WD. The columns without letters did not show significance\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8534257/v1/97e17283d9ce174a16df3db5.png"},{"id":103396107,"identity":"2b07b643-d30a-4cc5-9c9a-06b3ec5cfb28","added_by":"auto","created_at":"2026-02-25 08:46:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":407646,"visible":true,"origin":"","legend":"\u003cp\u003ePhotochemical efficiency parameters of maize plants under different soil and water conditions. Maximum quantum efficiency of photosystem II (Fv/Fm) in the morning (A) and afternoon (B); primary efficiency (Fv/F0) in the morning (C) and afternoon (D); and performance index (PI\u003csub\u003eABS\u003c/sub\u003e) in the morning (E) and afternoon (F). WW: well-watered; WD: water deficit; STE+RHIZO: sterile soil inoculated with \u003cem\u003eRhizoglomus clarum\u003c/em\u003e; NAT: natural soil; NAT+RHIZO: natural soil inoculated with \u003cem\u003eR. clarum\u003c/em\u003e. Data are means ± standard error (n = 4). Significant differences (ANOVA,\u0026nbsp; Tukey test, p \u0026lt; 0.05) are shown by letters. Capital letters compare treatments under WWand lower case letters compare treatments under WD. Asterisks indicate a significant difference between water regimes within the same treatment. The columns without letters did not show significance.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8534257/v1/8dae6b62306fa11ac6fc4c89.png"},{"id":106014027,"identity":"6b5f8801-9655-48c8-a9e7-42fbe0a09c6f","added_by":"auto","created_at":"2026-04-02 12:27:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3107780,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8534257/v1/c49a3523-192b-48e6-b799-ebc878eb96d9.pdf"},{"id":103507098,"identity":"09ddb04f-4138-4919-a023-3179acc0de7b","added_by":"auto","created_at":"2026-02-26 13:40:25","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":22998,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8534257/v1/8f829f2bb62da609b7ea14da.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rhizoglomus clarum inoculation enhances drought tolerance and photosynthetic performance of maize in sterile and natural soils","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWater deficit is a major abiotic stress limiting crop growth and productivity worldwide, with serious consequences on food security and the global economy (Irfan et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Several environmental factors, including reduced precipitation, high salinity, elevated temperatures, and low relative humidity, can increase evaporative demand and lead to water scarcity. In response, plants trigger physiological and biochemical responses (e.g., stomatal closure) to mitigate stress and survive. In addition, plants often rely on beneficial interactions with soil microorganisms to improve their stress tolerance. These associations are recognized as effective strategies to enhance tolerance to drought and nutrient limitations (Kobae \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gholinezhad et al.2020; Tang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Nie et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Given the complexity of plant physiological responses to drought, biological strategies that enhance stress tolerance through plant\u0026ndash;microbe interactions have gained increasing attention.\u003c/p\u003e \u003cp\u003eAmong these microorganisms, arbuscular mycorrhizal fungi (AMF) form an ancient and widespread symbiosis with plant roots (Nie et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) that plays a key role in drought tolerance. AMF improve water and nutrient uptake (Parniske \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kobae \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), regulate hormonal balance, modulate aquaporin expression, improve root hydraulic conductivity, and strengthen antioxidant defense systems (Sharma et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Abdelaal et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These combined effects enhance plant resilience to water deficit. In exchange, plants provide photosynthetically derived carbon to the fungal hyphae, which spread into the soil and access water and essential nutrients such as phosphorus, zinc, calcium, and manganese (Gomes J\u0026uacute;nior et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kobae \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Beyond improving nutrition, AMF also help to protect plants from various abiotic and biotic stresses (Brundrett and Tedersoo \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Moreira et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMaize, a crop highly sensitive to water stress during early development, benefits significantly from mycorrhizal associations. For example, \u003cem\u003eFunneliformis mosseae\u003c/em\u003e can delay the negative effects of drought, increase antioxidant enzyme activity, and reduce the accumulation of abscisic acid and reactive oxygen species (ROS). These effects result in improved growth and biomass (Ren et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bahraminia et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, most studies have focused on only a few AMF species, particularly \u003cem\u003eFunneliformis\u003c/em\u003e and \u003cem\u003eRhizophagus\u003c/em\u003e. As AMF species differ in colonization patterns, host specificity, and stress response mechanisms (Silva et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kozikova et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), species-specific studies are essential to understand and exploit their full potential in agriculture.\u003c/p\u003e \u003cp\u003eThis study evaluated how \u003cem\u003eRhizoglomus clarum\u003c/em\u003e inoculation affects maize physiological, biochemical, and photochemical traits under water deficit in sterile and natural soils. We hypothesize that \u003cem\u003eR. clarum\u003c/em\u003e improves drought tolerance by enhancing photochemical efficiency, supporting osmotic adjustment, and promoting vegetative growth.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area and plant material\u003c/h2\u003e \u003cp\u003eThe experiment was conducted in a greenhouse at the State University of Maranh\u0026atilde;o (UEMA), in S\u0026atilde;o Lu\u0026iacute;s, Maranh\u0026atilde;o, Brazil (2\u0026ordm;31\u0026rsquo;51\u0026rdquo; S, 44\u0026ordm;18\u0026rsquo;24\u0026rdquo; W; at 4 m.a.s.l.), from September to November 2023. The region has a tropical climate with a dry season (Aw) according to the K\u0026ouml;ppen\u0026ndash;Geiger classification (K\u0026ouml;pen-Geiger 2015).\u003c/p\u003e \u003cp\u003eEarly-cycle AG1051 (Seminis\u0026reg;) hybrid maize seeds were used. Soil was collected from the 0\u0026ndash;20 cm layer in a preserved vegetation area at the Farm School of UEMA and had the following chemical and physical characteristics: pH (CaCl\u003csub\u003e2\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;4.0; H\u0026thinsp;+\u0026thinsp;Al\u0026thinsp;=\u0026thinsp;41 cmol\u003csub\u003ec\u003c/sub\u003e dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e; Al\u003csup\u003e+\u0026thinsp;3\u003c/sup\u003e = 0 cmol\u003csub\u003ec\u003c/sub\u003e dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e; Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e = 1.0 cmol\u003csub\u003ec\u003c/sub\u003e dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e; Mg\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e = 4.0 cmol\u003csub\u003ec\u003c/sub\u003e dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e; K\u003csup\u003e+\u003c/sup\u003e = 4.6 cmol\u003csub\u003ec\u003c/sub\u003e dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e; P (Mehlich)\u0026thinsp;=\u0026thinsp;5.0 mg dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e; organic matter\u0026thinsp;=\u0026thinsp;11.0 g dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e; base saturation\u0026thinsp;=\u0026thinsp;19%; and texture\u0026thinsp;=\u0026thinsp;120 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e clay,30 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e silt, and 850 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sand (sandy loam soil). Fertilization followed the recommendation for pot experiments under controlled conditions (Novais et al. 1991). We applied 100 mg N, 300 mg P, and 150 mg K per kg of soil at planting.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInoculation of arbuscular mycorrhizal fungi (AMF)\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eRhizoglomus clarum\u003c/em\u003e (CNPAB-A05), obtained from the Embrapa Agrobiology germplasm bank (Serop\u0026eacute;dica, RJ) was used for AMF inoculations. To ensure successful colonization, we applied 1.43 g of inoculum (containing approximately 100 spores) next to the seeds at planting.\u003c/p\u003e\n\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cp\u003eA randomized block design was used, with four soil treatments: (1) sterile soil - control; (2) sterile soil inoculated with \u003cem\u003eR. clarum\u003c/em\u003e; (3) natural soil without inoculation; and (4) natural soil inoculated with \u003cem\u003eR. clarum\u003c/em\u003e. Each treatment was subjected to two water regimes: well-watered (WW) and water deficit (WD), resulting in eight treatment combinations with four replicates. Each experimental unit consisted of a 20 L pot. Five seeds were sown per pot and thinning was carried out on day 10, leaving one plant per pot. Soil for the sterile treatment was autoclaved at 120\u0026deg;C and 1.5 atm for 2 hours. The experiment lasted 51 days, ending at the V5-V6 developmental stage.\u003c/p\u003e \u003cp\u003eDuring the experiment, air temperature (\u0026deg;C), relative humidity (%), and photosynthetically active radiation (\u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were recorded every 15 minutesusing a WatchDog micro station (model 1000 Series, Spectrum Technologies, Inc., Illinois, USA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Based on temperature and relative humidity data, vapor pressure deficit (VPD\u003csub\u003eair\u003c/sub\u003e) was calculated according to Jones (1992) using the following formula :\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\text{F}\\text{o}\\text{r}\\text{m}\\text{u}\\text{l}\\text{a}\\:1.\\:{VPD}_{air}=0.61137\\times\\:\\text{exp}\\left(\\frac{\\begin{array}{c}17.502\\:\\times\\:\\:\\text{T}\\end{array}}{240.97+\\text{T}}\\right)\\times\\:\\:(1-\\frac{\\text{R}\\text{H}}{100}\\)\u003c/span\u003e \u003c/span\u003e)\u003c/p\u003e \u003cp\u003eWhere: VPD is the vapor pressure deficit (MPa); exp denotes the exponential function; T is the temperature (\u0026deg;C); and RH is the relative humidity (%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSoil moisture was maintained at 90% of field capacity (FC) through daily irrigation. Soil moisture levels were monitored using an RS485 TH-HMI043 sensor (ComWinTop, Mainland, China), following the methodology of Morales et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Readings were taken daily in all experimental units. Water restriction was imposed at 46 days after planting (DAP), between the V5 and V6 stages. This period is critical, as environmental stress can reduce the potential number of rows per ear (Magalh\u0026atilde;es and Dur\u0026atilde;es \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMorpho-anatomical measurements\u003c/h3\u003e\n\u003cp\u003eAt 51 DAP, leaf area (LA, cm\u003csup\u003e2\u003c/sup\u003e) was estimated according to the method of Radford (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1967\u003c/span\u003e), using the formula:\u003c/p\u003e \u003cp\u003eFormula 2. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:LA=K\\:(LL\\:\\times\\:\\:ML)\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eWhere LA is the leaf area (cm\u003csup\u003e2\u003c/sup\u003e), K is the constant (0.75), LL is the leaf length (cm), and ML is the maximum leaf width (cm).\u003c/p\u003e \u003cp\u003eOn the same day, plants were harvested and separated into leaves, stems, and roots. Each plant part was dried in a forced-air circulation oven at 70\u0026deg;C until constant weight. The dry mass of leaves (LDM), stems (SDM), and roots (RDM) was then determined.\u003c/p\u003e\n\u003ch3\u003eWater status\u003c/h3\u003e\n\u003cp\u003eRelative water content (RWC) was measured at 51 DAP using ten discs from a fully expanded leaf with a visible collar, ligule, and auricle. Fresh mass (FM, g) was determined immediately using an analytical balance. The discs were then rehydrated in deionized water for 24 h in the dark. Afterwards, excess surface water was removed with paper towels and turgid mass (TM, g) was recorded.\u003c/p\u003e \u003cp\u003eTo determine dry mass (DM, g), the discs were dried in a forced-air circulation oven at 65\u0026deg;C for 48 hours. RWC was calculated following Deng et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) using the formula:\u003c/p\u003e \u003cp\u003eFormula 3. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:RCW\\:\\left(\\%\\right)=\\frac{FM-TM}{TM-DM}\\:\\:\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eShoot water content (SW, g H\u003csub\u003e2\u003c/sub\u003eO) was estimated as the difference between the total fresh mass (leaves\u0026thinsp;+\u0026thinsp;stems) and total dry mass.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProline and total soluble carbohydrates\u003c/h2\u003e \u003cp\u003eProline was determined using 0.05 g of dry leaf tissue from the same samples used in the water status analysis. The extract was prepared with 2 mL of 3% sulfosalicylic acid. Proline content was determined according to the method of Bates et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1973\u003c/span\u003e), based on absorbance at 520 nm using a UV-5100 UV-Vis spectrophotometer (Shanghai Metash Instruments Co., Shanghai, China). The proline concentration (\u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM) values were calculated from a standard curve using L-proline.\u003c/p\u003e \u003cp\u003eTotal soluble carbohydrate (TSC) content was determined using 0.020 g of dry leaf mass, following the method of Dubois et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1956\u003c/span\u003e). Absorbance was read at 490 nm using the same spectrophotometer, and concentrations (mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM) were calculated from a standard glucose curve.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGreen intensity index and chlorophyll\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003efluorescence\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGreen intensity index (SPAD) was measured using a portable chlorophyll meter (model SPAD-502, Minolta, Japan). Measurements were taken on a fully expanded leaf between 8:00\u0026ndash;10:00 a.m. and 12:00\u0026ndash;2:00 p.m. during five days of water deficit. Ten readings per leaf were recorded, and the mean value per plant was calculated.\u003c/p\u003e \u003cp\u003eChlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence was measured on the same leaves using a portable fluorometer (FluorPen FP 110, Photon Systems Instruments, Dr\u0026aacute;sov, Czech Republic). Readings were taken from 8:00\u0026ndash;9:00 a.m. and 12:00\u0026ndash;1:00 p.m., after a 30-minute dark adaptation using leaf clips placed away from the midrib. A saturating light pulse (3000 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 650 nm, 1 second) was then applied to assess fluorescence transients, based on the OJIP test (Strasser et al. 1995). The following parameters were obtained: initial fluorescence (F\u003csub\u003e0\u003c/sub\u003e), variable fluorescence (Fv), maximum fluorescence (Fm), primary fluorescence (Fv/F\u003csub\u003e0\u003c/sub\u003e), maximum quantum efficiency of photosystem II (Fv/Fm), and photosynthetic performance index (PI\u003csub\u003eABS\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhotosynthetic pigments\u003c/h3\u003e\n\u003cp\u003eThe chlorophyll \u003cem\u003ea\u003c/em\u003e, chlorophyll b, and carotenoid contents were quantified from five leaf discs (0.6 cm\u0026sup2; each) collected from fresh tissues. The discs were placed in glass tubes wrapped in aluminum foil with 5 mL of 80% acetone and stored in the dark at 4\u0026deg;C for 72 hours. Absorbance of the extracts was measured at 480, 645, and 663 nm using a UV-5100 UV-Vis spectrophotometer (Shanghai Metash Instruments Co., Shanghai, China). Pigment concentrations were calculated using the equations of Lichtenthaler (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1987\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSpore extraction and morphological identification\u003c/h3\u003e\n\u003cp\u003eAMF glomerospores were extracted from 50 g of air-dried soil collected from the experimental treatments using the wet sieving and decanting method described by Gerdemann and Nicolson (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1963\u003c/span\u003e), followed by centrifugation in a sucrose solution according to Jenkins (1962). Spores were quantified on channeled plates under a stereomicroscope at 40\u0026times; magnification. For taxonomic identification, glomerospores were mounted on microscope slides with polyvinyl lacto-glycerol (PVLG) and a mixture of PVLG with Melzer\u0026rsquo;s reagent. They were examined under a compound microscope and identified based on morphological characteristics following Schenck and P\u0026eacute;rez (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) and using specialized online databases (INVAM, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://invam.caf.wvu.edu\u003c/span\u003e\u003cspan address=\"http://invam.caf.wvu.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eColonization rate\u003c/h2\u003e \u003cp\u003eAt the end of the water restriction period, maize roots were collected and thoroughly washed to remove adhering soil particles. Root samples were cleared in 5% (w/v) potassium hydroxide (KOH) and stained with trypan blue following the method of Phillips and Hayman (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). The stained roots were examined under a light microscope to confirm the presence of arbuscular mycorrhizal colonization. Plants were considered colonized when arbuscules, vesicles, spores, or hyphae were observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were tested for normality using the Shapiro-Wilk test. Variables meeting normality assumptions were subjected to analysis of variance (ANOVA), and the means were compared using Tukey test at a 5% significance level. Statistical analyses were performed in R software (version 3.4.1; R Core Team, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) using the \u0026ldquo;ExpDes.pt\u0026rdquo; (Ferreira et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and \u0026ldquo;factoextra\u0026rdquo; (Kassambara \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) packages.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of AMF species and colonization rate\u003c/h2\u003e \u003cp\u003eEight AMF species were recovered from the natural soil used in the experiment. Based on morphological characteristics, the following species were identified: \u003cem\u003eAcaulospora morrowiae\u003c/em\u003e Spain \u0026amp; N.C. Schenck, \u003cem\u003eAcaulospora scrobiculata\u003c/em\u003e Trappe, \u003cem\u003eAcaulospora foveate\u003c/em\u003e St\u0026uuml;rmer \u0026amp; Morton, \u003cem\u003eAmbispora appendicula\u003c/em\u003e Spain, Sieverd. \u0026amp; N.C. Schenck) C. Walker, and four unidentified Glomus morphotypes (\u003cem\u003eGlomus\u003c/em\u003e sp1, \u003cem\u003eGlomus\u003c/em\u003e sp2, \u003cem\u003eGlomus\u003c/em\u003e sp3 [\u003cem\u003eglomerulatum\u003c/em\u003e like], and \u003cem\u003eGlomus\u003c/em\u003e sp4).\u003c/p\u003e \u003cp\u003eRoot colonization was significantly affected by soil condition and AMF inoculation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Under water deficit, NAT\u0026thinsp;+\u0026thinsp;RHIZO treatment showed the highest colonization rate (40.6%), representing a 1.92-fold increase compared to STE\u0026thinsp;+\u0026thinsp;RHIZO and a 1.41-fold increase compared to NAT alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLeaf area and dry biomass\u003c/h2\u003e \u003cp\u003eA significant difference in leaf area (LA) was observed among treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.03). Under well-watered conditions, the NAT\u0026thinsp;+\u0026thinsp;RHIZO treatment showed the highest mean LA (736.3 cm\u0026sup2;). Under water deficit, NAT\u0026thinsp;+\u0026thinsp;RHIZO had a 36.8% increase in LA compared to the control (WD_STE) (Fig.\u0026nbsp;3A).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;3\u003c/b\u003e Growth parameters of maize plants under different soil and water conditions. (A) Leaf area, (B) leaf dry mass, (C) stem dry mass, and (D) root dry mass. WW: well-watered; WD: water deficit; STE\u0026thinsp;+\u0026thinsp;RHIZO: sterile soil inoculated with \u003cem\u003eRhizoglomus clarum\u003c/em\u003e; NAT: natural soil; NAT\u0026thinsp;+\u0026thinsp;RHIZO: natural soil inoculated with \u003cem\u003eR. clarum\u003c/em\u003e. Data are means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (n\u0026thinsp;=\u0026thinsp;4). Significant differences (ANOVA, Tukey test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) are shown by letters. Capital letters compare treatments under WW and lower case letters compare treatments under WD. Asterisks indicate a significant difference between water regimes within the same treatment. The columns without letters did not show significance\u003c/p\u003e \u003cp\u003eLeaf dry mass (LDM), stem dry mass (SDM), and root dry mass (RDM) differed among treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00) (Fig.\u0026nbsp;3B-D). Under water deficit, LDM in the NAT\u0026thinsp;+\u0026thinsp;RHIZO treatment was 45.4% higher than in the control STE. Under well-watered conditions, NAT\u0026thinsp;+\u0026thinsp;RHIZO also showed the highest LDM with 43.7% increase compared to STE (Fig.\u0026nbsp;3B). For SDM, NAT\u0026thinsp;+\u0026thinsp;RHIZO resulted in 100% increase over the control STE under water deficit and 63.16% increase in well-watered conditions (Fig.\u0026nbsp;3C). RDM was 43.7% higher in STE\u0026thinsp;+\u0026thinsp;RHIZO when compared to NAT under water deficit (Fig.\u0026nbsp;3D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eWater status\u003c/h2\u003e \u003cp\u003eRelative water content (RWC) and shoot water (SW) differed significantly between the water regimes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Under well-watered conditions, SW was higher in the NAT\u0026thinsp;+\u0026thinsp;RHIZO treatment compared to STE and STE\u0026thinsp;+\u0026thinsp;RHIZO, with icreases of 39.6% and 38.7%, respectively. In contrast, we observed no differences in SW among treatments under water deficit conditions (p\u0026thinsp;\u0026gt;\u0026thinsp;0.075) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTotal soluble carbohydrate, proline, and photosynthetic pigment contents\u003c/h2\u003e \u003cp\u003eTotal soluble carbohydrate (TSC) content differed between the water regimes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In the STE\u0026thinsp;+\u0026thinsp;RHIZO treatment, TSC was 114.1% higher under well-watered conditions than under water deficit RHIZO. We observed a similar trend in the NAT\u0026thinsp;+\u0026thinsp;RHIZO treatment, with a 95.3% increase under irrigation RHIZO. Proline content varied among treatments only under water deficit (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with the NAT treatment showing the highest levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Concerning the photosynthetic pigments, carotenoid content differed significantly only among treatments under well-watered conditions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.03), with the highest value observed in NAT (0.818 \u0026micro;mol g⁻\u0026sup1;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In contrast, chlorophyll \u003cem\u003ea\u003c/em\u003e, chlorophyll b, and total chlorophyll contents were similar across treatments (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStomatal density\u003c/h2\u003e \u003cp\u003eAbaxial stomatal density differed among treatments under both well-watered (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and water-deficit (p\u0026thinsp;\u0026lt;\u0026thinsp;0.03) conditions. Under water deficit, STE had the lowest mean density (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). For the adaxial surface, significant differences were observed only under well-watered conditions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with the highest mean in the NAT\u0026thinsp;+\u0026thinsp;RHIZO treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Concerning stomatal functionality, only the abaxial surface showed a statistical difference under water-deficit conditions (p\u0026thinsp;\u0026gt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eChlorophyll\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003efluorescence\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe maximum quantum yield of photosystem II (Fv/Fm) differed between water regimes in the morning (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). NAT\u0026thinsp;+\u0026thinsp;RHIZO and STE\u0026thinsp;+\u0026thinsp;RHIZO treatments showed the highest Fv/Fm values in well-watered plants, with the former standing out (i.e., 50% higher than the control) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). STE\u0026thinsp;+\u0026thinsp;RHIZO also showed a significant increase of 14.7% relative to the control. In the afternoon, significant differences were observed only under water deficit conditions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In the NAT\u0026thinsp;+\u0026thinsp;RHIZO and WD_STE\u0026thinsp;+\u0026thinsp;RHIZO treatments had Fv/Fm values 52.9% and 56.6% higher, respectively, than STE (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrimary fluorescence efficiency (Fv/F\u003csub\u003eo\u003c/sub\u003e) differed among treatments only under water-deficit, both in the morning and afternoon (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). In the afternoon, Fv/F\u003csub\u003eo\u003c/sub\u003e was higher in the STE\u0026thinsp;+\u0026thinsp;RHIZO and NAT\u0026thinsp;+\u0026thinsp;RHIZO treatments, with the latter showing a 1.26-fold increase compared to STE (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003ePerformance index (PI\u003csub\u003eABS\u003c/sub\u003e) differed among water-deficit treatments in the morning (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eE) and well-watered treatments in the afternoon (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). WD_NAT had the highest mean and NAT\u0026thinsp;+\u0026thinsp;RIZHO also differed between water regimes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). In the afternoon, WW_NAT\u0026thinsp;+\u0026thinsp;RHIZO had the highest mean (43.9% increase compared to WW_STE) and the only treatment showing differences between water regimes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Green intensity index (SPAD) values were similar among treatments and water regimes (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Supplementary Table S2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDrought negatively affects maize growth by reducing water availability, limiting leaf and root development, and decreasing photosynthetic rate and water use efficiency, ultimately impairing plant productivity (Avramova et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this context, AMF have emerged as beneficial symbionts that help plants cope with water stress. Here, the successful colonization of maize roots by \u003cem\u003eR. clarum\u003c/em\u003e confirmed the establishment of a functional mycorrhizal symbiosis. Although drought reduces the colonization rate (Begum et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), inoculation of \u003cem\u003eR. clarum\u003c/em\u003e in natural soil increased the mycorrhizal colonization rate, resulting in similar levels between irrigated and water-stressed treatments.\u003c/p\u003e \u003cp\u003eThis association led to improvements in key growth and physiological traits. Inoculated plants in natural soil had increased leaf area under both well-watered and water-deficit conditions (Fig.\u0026nbsp;3A). A larger leaf area contributes to improved light interception and photosynthetic capacity (Luiz et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Pinheiro et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which are critical for maintaining growth under drought. Similar increases in leaf area have been observed in other studies involving maize and AMF under stress, reinforcing their positive role in leaf development (Agbodjato et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInoculation with \u003cem\u003eR. clarum\u003c/em\u003e significantly increased the dry biomass of maize shoots, stems, and roots (Fig.\u0026nbsp;3B-D). AMF can enhance plant growth by improving water and nutrient uptake through their hyphae, stimulating root growth and mitigating drought (Tian et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ren et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The stronger biomass response in NAT\u0026thinsp;+\u0026thinsp;RHIZO compared to STE\u0026thinsp;+\u0026thinsp;RHIZO suggests a synergistic interaction between the inoculated \u003cem\u003eR. clarum\u003c/em\u003e and native microbial communities. Indigenous AMF and beneficial rhizobacteria may facilitate colonization, enhance hyphal proliferation, or contribute complementary drought-mitigation mechanisms, a pattern increasingly recognized in studies on AMF functioning in natural soils (Sharma et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Abdelaal et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Similar benefits of AMF have been reported under other abiotic stress conditions, such as salinity in maize (Xu, Lu and Tong \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eContrary to previous studies reporting higher relative water content in AMF-inoculated plants (Zhu et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Santos et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we observed no significant differences under our experimental conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Relative water content is an important indicator of plant water status and is closely linked to photosynthesis, biomass accumulation, and productivity (Ashraf et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast, shoot water content was positively influenced by \u003cem\u003eR. clarum\u003c/em\u003e inoculation, particularly under well-watered conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Higher shoot water in inoculated plants suggests that AMF may help maintain leaf hydration. Similar results were reported by Ait-El-Mokhtar et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who observed improved leaf water content under drought. This improvement is likely related to increased hydraulic conductivity and more efficient water absorption mediated by AMF.\u003c/p\u003e \u003cp\u003eBoth proline and total carbohydrate contents were higher in AMF-inoculated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B), indicating enhanced osmotic adjustment. Proline helps plants maintain osmotic homeostasis, stabilizes proteins and membranes, and mitigates oxidative damage under water stress (Laxa et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sadak et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Soluble carbohydrates also contribute to stress tolerance by acting as antioxidants and regulating reactive oxygen species (ROS) signaling (Begum et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The accumulation of these compatible solutes likely contributed to maintaining water status and improving plant resilience under drought, consistent with findings reported by Begum et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Carotenoid levels were higher in plants grown in natural soil (NAT) compared to those in sterile soil STE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), suggesting that native communities of arbuscular mycorrhizal fungi, along with the existing soil microbiota to photoprotection. Carotenoids protect plants against oxidative stress by quenching ROS and dissipating excess light energy, thus preventing photoinhibition (Hazrati et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Our results align with studies showing that native AMF can enhance antioxidant capacity through functional complementarity (Begum et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ye et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite evidence that drought can reduce chlorophyll content (Avramova et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and that AMF may improve green intensity index under stress (Yan et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), no significant differences in chlorophyll contents or SPAD values were observed among treatments in this study. Chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence is widely used to assess the efficiency of photosystem II (PSII) and plant responses to stress (Murchie and Lawson \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ye, Wang and Li \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Drought reduced PSII efficiency as indicated by declines in Fv/Fm, Fv/F\u003csub\u003eo\u003c/sub\u003e, and performance index (PI\u003csub\u003eABS\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The observed Fv/Fm values were below the typical range for healthy plants (0.75\u0026ndash;0.85), confirming the severity of stress (Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, inoculation with \u003cem\u003eR. clarum\u003c/em\u003e reduced the magnitude of this decrease, leading to higher Fv/Fm values ​​compared to non-inoculated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results suggest that AMF symbiosis attenuated photoinhibition and contributed to the partial maintenance of photosynthetic activity, likely by enhancing water uptake, stabilizing membrane integrity, and reducing oxidative stress (Tang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rasouli et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Similar protective effects have been reported in maize and other crops under drought and heat stress (Zhu et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Porcel et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Maia et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaken together, this study demonstrates that \u003cem\u003eR. clarum\u003c/em\u003e inoculation enhances maize drought tolerance through coordinated improvements in growth, water status, osmotic adjustment, and photochemical efficiency. The stronger effects observed in natural soil suggest a synergistic interaction between \u003cem\u003eR. clarum\u003c/em\u003e and indigenous microbial communities, reinforcing the value of AMF consortia in field conditions. AMF-inoculated plants maintained higher biomass, accumulated more compatible solutes, and showed improved photosystem II performance under stress, supporting the view that AMF act as holistic modulators of plant resilience. These results contribute to a growing of evidence that AMF can mitigate abiotic stress beyond nutrient uptake, with implications for sustainable crop production under water-limited conditions (Tang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Abdelaal et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough our study did not directly assess molecular mechanisms, previous research suggests that AMF can enhance drought tolerance by increasing root hydraulic conductivity, modulating aquaporin expression, and regulating hormonal pathways such as abscisic acid and jasmonates (Tang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ye et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These adjustments collectively improve water status and carbon assimilation under limited moisture conditions. Future research integrating transcriptomics and root hydraulic analyses could elucidate the specific pathways by which \u003cem\u003eR. clarum\u003c/em\u003e promotes adaptation to drought in natural soils.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study underscores the potential of \u003cem\u003eR. clarum\u003c/em\u003e as a microbial tool for enhancing maize resilience under drought. Beyond confirming the functional role of AMF, our findings point to the importance of species-specific interactions and soil context in shaping plant responses to water stress. The synergistic effects observed in natural soil highlight the value of local microbial communities to optimize inoculant performance. Integrating AMF-based inoculants into agricultural systems may offer a scalable, low-input, and sustainable approach to improve crop stability under increasingly variable climate conditions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eArbuscular mycorrhizal fungi\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eChl a\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChlorophyll \u003cem\u003ea\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eChl b\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChlorophyll \u003cem\u003eb\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDays after planting\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDry mass\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eF₀\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMinimum fluorescence yield\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eField capacity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFresh mass\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFm\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMaximum fluorescence yield\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFv/F₀\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInitial photochemical efficiency of PSII\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFv/Fm\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMaximum quantum efficiency of PSII\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLeaf area\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLDM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLeaf dry mass\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNAT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNatural soil\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNAT\u0026thinsp;+\u0026thinsp;RHIZO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNatural soil\u0026thinsp;+\u0026thinsp;\u003cem\u003eRhizoglomus clarum\u003c/em\u003e inoculation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePI\u003csub\u003eABS\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePerformance index based on absorption\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePSII\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhotosystem II\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRDM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRoot dry mass\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRHIZO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eRhizoglomus clarum\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRWC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRelative water content\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStem dry mass\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSoil and Plant Analyzer Development Index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSterile soil\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTE\u0026thinsp;+\u0026thinsp;RHIZO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSterile soil\u0026thinsp;+\u0026thinsp;\u003cem\u003eRhizoglomus clarum\u003c/em\u003e inoculation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eShoot water\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTurgor mass\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTotal soluble carbohydrate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVPD\u003csub\u003eair\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVapor pressure deficit of air\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWater deficit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWell\u0026ndash;watered.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eTMF and CPN conceived the study and supervised the project. TMF, NBC, SOMJ, CPN, and MRSAL designed and performed experiments. MRSAL and NBC wrote the manuscript. MRSAL, NBC, PCS, DBC, SOMJ, and JRA assisted in performing the experiments. TRC, FOR, WPR, PHAC, ION, CPN, FAMAF, and TMF review and editing manuscript. MRSAL and NBC Review and editing finally for submission. All authors read and approved the final\u0026nbsp;manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis study was carried out support of the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brasil (CAPES) - Finance Code 001. The authors also acknowledge the support of the National Council for Scientific and Technological Development (CNPq): PQ 312653/2025-5 awarded to Tiago M. Ferraz, PQ 307349/2023-3 awarded to Thais R. Corr\u0026ecirc;a, PQ 305522/2025-6 awarded to Fabr\u0026iacute;cio de O. Reis, and PQ 306442/2025-6 awarded to F\u0026aacute;bio A. M. de A. Figueiredo.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdelaal K, Alaskar A, Hafez Y (2024) Effect of arbuscular mycorrhizal fungi on physiological, bio-chemical and yield characters of wheat plants (\u003cem\u003eTriticum aestivum L\u003c/em\u003e.) under drought stress conditions. BMC Plant Biol 24:1119. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12870-024-05824-9\u003c/span\u003e\u003cspan address=\"10.1186/s12870-024-05824-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgbodjato NA et al (2022) Formulation of biostimulants based on arbuscular mycorrhizal fungi for maize growth and yield. 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Plant soil 346:189\u0026ndash;199. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11104-011-0809-8\u003c/span\u003e\u003cspan address=\"10.1007/s11104-011-0809-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"Photochemical efficiency, Rhizoglomus clarum, water restriction, Zea mays L","lastPublishedDoi":"10.21203/rs.3.rs-8534257/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8534257/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDrought is a major constraint on maize production worldwide, particularly in tropical regions where climate variability is intensifying. Arbuscular mycorrhizal fungi (AMF) have emerged as beneficial symbionts enhancing plant resilience to drought by improving water uptake, nutrient acquisition, and photosynthetic performance. This study evaluated the effects of \u003cem\u003eRhizoglomus clarum\u003c/em\u003e inoculation on maize growth, water status, osmotic adjustment, and chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence under well-watered (WW) and water-deficit (WD) conditions in sterile and natural soils. The experiment was conducted in a greenhouse using a randomized complete block design in a 4 \u0026times; 2 factorial scheme (soil treatment \u0026times; water regime), with four replicates. Drought significantly reduced leaf area, shoot and root biomass, and water status. However, \u003cem\u003eR. clarum\u003c/em\u003e inoculation attenuated these effects, increasing leaf dry mass by up to 45% and stem dry mass by 100% in under WD. Inoculated plants also showed higher photochemical efficiency (Fv/Fm and PI\u003csub\u003eABS\u003c/sub\u003e) under both water regimes. The strongest responses were observed in natural soil, suggesting synergistic interactions between \u003cem\u003eR. clarum\u003c/em\u003e and indigenous microbiota. These results demonstrate that \u003cem\u003eR. clarum\u003c/em\u003e enhances maize drought tolerance through coordinated morphological, physiological, and photochemical adjustments. This highlights the potential of species-specific AMF inoculation as a sustainable strategy to improve maize performance under water-limited conditions.\u003c/p\u003e","manuscriptTitle":"Rhizoglomus clarum inoculation enhances drought tolerance and photosynthetic performance of maize in sterile and natural soils","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-25 08:46:36","doi":"10.21203/rs.3.rs-8534257/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":"7b43192a-9e08-457b-84b2-e1eba71087e2","owner":[],"postedDate":"February 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-02T12:26:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-25 08:46:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8534257","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8534257","identity":"rs-8534257","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}