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Article

Rhizosphere Growth-Promoting Fungi of Healthy Nicotiana tabacum L.: A Systematic Approach to Boosting Plant Growth and Drought Resistance

by
Quanyu Yin
1,
Zhao Feng
1,
Zhichao Ren
1,
Ao Li
1,
Amit Jaisi
2,3 and
Mengquan Yang
1,*
1
National Tobacco Cultivation, Physiology and Biochemistry Research Center, College of Tobacco Science, Henan Agricultural University, Zhengzhou 450046, China
2
School of Pharmacy, Walailak University, Thasala, Nakhon Si Thammarat 80160, Thailand
3
Biomass and Oil Palm Center of Excellence, Walailak University, Thasala, Nakhon Si Thammarat 80160, Thailand
*
Author to whom correspondence should be addressed.
Microorganisms 2025, 13(3), 543; https://doi.org/10.3390/microorganisms13030543
Submission received: 2 January 2025 / Revised: 9 February 2025 / Accepted: 25 February 2025 / Published: 27 February 2025
(This article belongs to the Section Plant Microbe Interactions)
Browse Figures
Versions Notes

Abstract

:
Drought, exacerbated by global warming, poses a significant threat to crop growth and productivity. This study identified a strain of Trichoderma harzianum from the rhizosphere of healthy Nicotiana tabacum L. plants and evaluated its role in enhancing drought tolerance. The isolated strain effectively colonized plant roots and promoted the growth of N. tabacum L. To investigate its potential, T. harzianum was inoculated into plants under varying drought conditions, and its impact on growth, physiological responses, and drought resilience was assessed. Comprehensive analyses of agronomic traits, physiological parameters, enzyme activities, photosynthetic performance, osmoprotectant levels, and membrane lipid peroxidation revealed that T. harzianum inoculation (light drought with T. harzianum, moderate drought with T. harzianum, and severe drought with T. harzianum treatments) systematically improved plant development and drought resistance. These findings provide valuable insights and lay a foundation for developing innovative biofertilizers to enhance crop drought tolerance and sustainability.

1. Introduction

As the greenhouse effect intensifies, global temperatures continue to rise which is leading to an increased frequency of droughts [1]. This, in turn, causes significant reductions in crop yields, and in extreme cases, total crop failure. Water, being a crucial factor in plant growth and development, is indispensable. Its absence severely impacts the growth, yield, and quality of crops [2]. Consequently, studying plant responses to drought and developing new biofertilizers to enhance plant drought resistance have become prominent research topics [3,4].
In recent years, microbial inoculation has proven to be an effective method for promoting plant growth [5,6]. It can improve the soil’s microbial community structure, enhance the uptake of nutrients by plants, and increase their resistance to both biotic and abiotic stresses, thereby ensuring healthy growth [7,8,9,10]. Although the application of microbial inoculation in agriculture is widely recognized, its comprehensive effects on plants have not yet been fully explored. Recently, an increasing number of bacteria and fungi, such as Bacillus [11,12], Pseudomonas [13,14], arbuscular mycorrhizal fungi [12,15,16], and Trichoderma [17,18], have been shown to promote plant growth.
Nicotiana tabacum L., a widely cultivated economic crop, also serves as an important model organism in plant research [19]. Recently, N. tabacum L. has been developed as a chassis for synthetic biology, particularly for the production of natural products and antibodies [20,21,22]. Current research on N. tabacum L. primarily focuses on responses to biotic stressors (pathogens and insects) and abiotic stressors (UV radiation, drought, salinity, and waterlogging) [19,23,24]. However, studies on enhancing N. tabacum L.’s resilience to abiotic stress through microbial inoculation remain limited [25]. Therefore, it is crucial to investigate the changes in N. tabacum L. to elucidate the mechanisms by which Trichoderma. harzianum inoculation enhances drought resistance in this species.
This study isolated a strain of T. harzianum from the rhizosphere soil of healthy N. tabacum L. plants and evaluated its growth promoting effects on N. tabacum L. seedlings. Further experiments involving the inoculation of N. tabacum L. with T. harzianum demonstrated significant improvements in growth under drought stress conditions. Comprehensive analyses of plant phenotypes and physiological and biochemical indicators elucidated the mechanisms by which this plant growth-promoting fungus enhances drought resistance. This research developed a microbial inoculant capable of improving the drought tolerance of N. tabacum L., offering a potential solution to water scarcity issues exacerbated by global climate change.

2. Materials and Methods

2.1. Trichoderma Harzianum Isolation and Drought Treatment

Soil samples from the rhizosphere of healthy N. tabacum L. plants in a field affected by Phytophthora nicotianae (tobacco black shank disease) were collected to isolate Trichoderma strains. The strains were isolated from the soil samples using the dilution plating method and cultured on a PDA medium at 28 °C [26]. The growth promoting effects of the isolated Trichoderma strains on N. tabacum L. seedlings were evaluated. The colonization of the obtained T. harzianum in the plants was observed using acid fuchsin and fast green staining. The T. harzianum used in this study was isolated from the N. tabacum L. rhizosphere soil which was deposited at the China General Microbiological Culture Collection (CGMCC No. 23294).
The study was conducted using Nicotiana tabacum cultivar K362, and seeds provided by the College of Tobacco Science at Henan Agricultural University. Seedlings were germinated from seeds in controlled conditions and subsequently transplanted. To systematically study the mechanisms, a total of 180 well-grown plants at the same growth stage were selected for drought and T. harzianum treatments. Plants were divided into 6 groups, with 30 plants in each group, as follows: light drought (LD), light drought with T. harzianum (LDTh), moderate drought (MD), moderate drought with T. harzianum (MDTh), severe drought (SD), and severe drought with T. harzianum (SDTh) (Figure 1). Soil moisture levels were maintained as follows: CK (control): 70% of field capacity; LD (light drought): 60% of field capacity; MD (moderate drought): 50% of field capacity; SD (severe drought): 40% of field capacity. Soil moisture levels were monitored periodically to ensure consistency.
After 2 days of drought and T. harzianum root drenching treatment, the first assessment of plant growth was conducted, and samples were collected for further analyses. Sampling was performed every five days, with a total of four collections labeled as the 1st, 2nd, 3rd, and 4th. The collected samples were rapidly frozen in liquid nitrogen (N) and stored at −80 °C for subsequent physiological and biochemical analyses.

2.2. Agronomic Traits

Agronomic traits of the N. tabacum L. plants were measured using a tape measure, following the guidelines outlined in YC/T 142-2010, ’Methods for Survey and Measurement of Tobacco Agronomic Traits’ [27]. The parameters investigated included plant height, stem grith, maximum leaf length, maximum leaf width, and the number of effective leaves.

2.3. Root Development

The entire root system was carefully cleaned and scanned for appearance and morphology using an EPSON Expression 12000 XL plant root scanner (Epson, Nagano, Japan). The root system was then analyzed using WinRHIZO root analysis software (Regent Instruments, Quebec, QC, Canada, version Pro2007d) to determine the total root length, projection area, root surface area, root volume, root diameter, root tip number, branch number, and number of connections.

2.4. Relative Water Content of Leaves

A leaf sample (the third leaf from the top) was taken to measure the relative moisture content using the drying method [28]. Fresh N. tabacum L. leaves were first weighed, then saturated with water and weighed again. Finally, the leaves were dried and weighed. The relative water content was calculated as the percentage of the fresh leaf water content in relation to the saturated water content.

2.5. Root Activity

Root activity was measured using the TTC method [29]. During the measurement, 0.5 g of the root system was placed in a test tube with 10 mL of an equal mixture of 0.4% TTC solution and phosphate buffer. The mixture was incubated at 37 °C for 1 h in the dark, followed by the addition of 2 mL of 1 mol/L sulfuric acid to stop the reaction. The root was then removed, and surface moisture was absorbed. The root was placed in a test tube with 10 mL of methanol solution and kept in the dark at 37 °C for 2.5 h. A spectrophotometer (Spectronic 20 Genesys, Spectronic Instruments, Rochester, NY, USA) was used to measure the color at 485 nm, and the reduction amount of tetrazole was calculated.

2.6. Photosynthetic Parameters

The net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate were measured using a Li-6400 portable photosynthetic instrument (LI-COR Inc., Lincoln, NE, USA) between 9:00 a.m. and 11:30 a.m.

2.7. Photosynthetic Pigments

Following Wellburn’s method [30], the main veins were removed from the N. tabacum L. leaves, and 0.5 g of the leaves were weighed and placed in a 50 mL centrifuge tube. Then, 25 mL of 95% ethanol was added, and the tube was stored in a sealed, dark place for 24–36 h. A spectrophotometer (Spectronic 20 Genesys, Spectronic Instruments, Rochester, NY, USA) was used to measure the optical density (OD) at 665 nm, 649 nm, and 470 nm to determine the concentrations of chlorophyll a, chlorophyll b, and carotenoids, respectively. The concentrations were calculated using the formulas: Ca = 13.95D665 − 6.88D649; Cb = 24.96D649 − 7.32D665. The pigment content (mg/g) was then determined using the following formula: pigment content (mg/g) = (pigment concentration (mg/L) × extraction solution volume (mL) × dilution ratio)/sample mass (g).

2.8. Osmoprotectants

The same part of the leaf was frozen with liquid N and stored in a refrigerator at −80 °C. The contents of proline, soluble sugar, and soluble protein were determined using the Solarbio reagent kit, following the manufacturer’s protocol.

2.9. Leaf Protective Enzyme Activity

The same part of the leaf was frozen with liquid N and stored at −80 °C. The activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and ascorbate peroxidase (APX) were measured using the Solarbio reagent kit.

2.10. Membrane Lipid Peroxidation Index

The same part of the leaf was frozen with liquid N and stored at −80 °C. The contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) were determined using the Solarbio reagent kit, following the manufacturer’s protocol.

2.11. Nitrate Reductase Activity

After being rapidly frozen in liquid N, the samples were stored at −80 °C. The nitrate reductase (NR) activity was measured using a UV–visible spectrophotometer, with the NR activity assay kit purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

2.12. Data Processing

For the measurement of agronomic traits, 10 replications were performed, while physiological parameters were measured with 3 replications. The data were analyzed for statistical significance using one-way analysis of variance (ANOVA). A significance level of p < 0.05 was considered statistically significant.
The data were processed using Microsoft Excel 2019 (Microsoft Corporation, Redmond, WA, USA) and IBM SPSS Statistics 26.0 (IBM Corporation, Armonk, NY, USA) software for analysis of variance, and the significance of the data differences (lowercase letters on the bars in the figures) was tested. OriginPro 2021 (OriginLab Corporation, Northampton, MA, USA) was used for plotting.

3. Results

3.1. Plant Growth-Promoting Fungi Isolation and Its Effects Against Drought

Rhizosphere soil from healthy N. tabacum L. plants was collected from fields infected by tobacco black shank disease to isolate plant growth-promoting fungi (Figure 2A). A fungus capable of growing on the PDA medium was successfully isolated, identified as T. harzianum (Figure 2B), and deposited into the China General Microbiological Culture Collection (CGMCC No. 23294). Inoculation of N. tabacum L. seedlings with T. harzianum showed a significant growth-promoting effect under drought conditions (Figure 2C). Staining and microscopic observation of the inoculated N. tabacum L. roots revealed that T. harzianum (green) successfully colonized the roots (red), thereby enhancing plant growth and development (Figure 2D and Figure S4).

3.2. Agronomic Traits of N. tabacum L. After T. harzianum Inoculation During DS Treatment

Abiotic stress often significantly affects plant phenotypes. Therefore, agronomic traits were systematically recorded and analyzed across various treatments. A comparison of T. harzianum inoculation effects under varying drought conditions revealed that the inoculation significantly promoted plant growth and enhanced drought tolerance across light, moderate, and severe drought levels (Figure S1). Measurements revealed that under drought stress, T. harzianum inoculation had a notable impact on plant height (Figure 3A), stem grith (Figure 3B), maximum leaf length (Figure 3C), maximum leaf width (Figure 3D), and the number of effective leaves (Figure 3E). The metrics for T. harzianum treatments (LDTh, MDTh, SDTh) were consistently higher than those for the corresponding drought only groups (LD, MD, SD). Specifically, plant height increased by 10.30%, 9.64%, and 15.36%; stem girth by 16.52%, 8.74%, and 7.69%; maximum leaf length by 8.78%, 4.27%, and 5.74%; maximum leaf width by 8.40%, 13.03%, and 9.51%; and the number of effective leaves by 20.00%, 21.05%, and 14.71%, respectively (Figure 3).

3.3. Root System of N. tabacum L. After T. harzianum Inoculation During DS Treatment

Roots, as essential organs for water and nutrient absorption, play a crucial role in providing plants with the necessary substances for growth. Observation of the root systems revealed that the roots of plants inoculated with T. harzianum were generally better developed than those of the non-inoculated plants (Figure S2). An assessment of root system parameters showed that T. harzianum inoculation under drought stress significantly affected total root length (Figure 4A), projection area (Figure 4B), root surface area (Figure 4C), root volume (Figure 4D), root diameter (Figure 4E), root tip number (Figure 4F), branch number (Figure 4G), root connectivity (Figure 4H), and root activity (Figure 4I). Compared to the corresponding drought-only treatments (LD, MD, and SD), all indicators in the T. harzianum-inoculated treatments (LDTh, MDTh, and SDTh) were consistently higher. Specifically, total root length increased by 17.90%, 29.87%, and 47.68%; root projection area by 39.86%, 62.97%, and 32.41%; root surface area by 19.68%, 24.40%, and 41.19%; root volume by 101.36%, 166.89%, and 55.22%; root diameter by 28.30%, 21.38%, and 30.46%; root tip number by 73.58%, 61.72%, and 60.67%; branch number by 30.07%, 24.32%, 39.66%; root connectivity by 30.43%, 36.10%, and 30.72%; root activity by 36.69%, 33.63%, 16.75%.

3.4. Photosynthetic Parameters of N. tabacum L. After T. harzianum Inoculation During DS Treatment

Photosynthesis is one of the most critical chemical reactions on Earth and is among the physiological processes in green plants most sensitive to drought. Measurements of photosynthetic parameters showed that inoculation with T. harzianum under drought stress significantly enhanced net photosynthetic rate (Figure 5A), stomatal conductance (Figure 5B), transpiration rate (Figure 5C), intercellular CO2 concentration (Figure 5D), and leaf relative water content (Figure 5E). In all cases, the indicators in the Trichoderma-treated groups (LDTh, MDTh, and SDTh) were higher than those in the corresponding drought-only groups (LD, MD, and SD). Specifically, the net photosynthetic rate increased by 48.89%, 101.56%, and 52.75%; stomatal conductance by 82.03%, 43.72%, and 288.78%; intercellular CO2 concentration by 20.01%, 36.79%, and 43.12%; transpiration rate by 34.29%, 33.93%, and 152.71%; and leaf relative water content by 3.60%, 5.43%, and 4.74%.

3.5. Photosynthetic Pigments of N. tabacum L. After T. harzianum Inoculation During DS Treatment

After measuring photosynthetic parameters, the associated pigments were also assessed. Inoculation with T. harzianum (LDTh, MDTh, and SDTh) resulted in higher levels of both chlorophyll a (Figure 6A) and chlorophyll b (Figure 6B) compared to the corresponding drought treatments (LD, MD, and SD). Chlorophyll a content increased by 5.79%, 5.00%, and 81.73%, while chlorophyll b content increased by 17.23%, 44.64%, and 32.30%. The chlorophyll a to b ratio ranged from 0.5 to 2.5 and was significantly higher under severe drought (SD) conditions during the first and third sampling periods (Figure 6C).

3.6. Osmoprotectants of N. tabacum L. After T. harzianum Inoculation During DS Treatment

Plants often respond to abiotic stress by altering the levels of osmotic regulators. Therefore, we measured the contents of soluble sugar (Figure 7A), soluble protein (Figure 7B), and proline (Figure 7C). Inoculation with T. harzianum (LDTh, MDTh, and SDTh) resulted in higher levels of these substances compared to the corresponding drought treatments (LD, MD, and SD). Specifically, soluble sugar content increased by 51.38%, 7.80%, and 19.62%; soluble protein content increased by 22.39%, 33.11%, and 24.81%; and proline content increased by 10.01%, 15.28%, and 31.00%.

3.7. Membrane Lipid Peroxidation Index of N. tabacum L. After T. harzianum Inoculation During DS Treatment

The membrane lipid peroxidation index, mainly H2O2 (hydrogen peroxide) and MDA (malondialdehyde) contents, reflect the oxidative stress experienced by plants. Measurements revealed that H2O2 (Figure 8A) and MDA (Figure 8B) contents in Trichoderma-treated groups (LDTh, MDTh, and SDTh) were lower than those in the corresponding drought-treated groups (LD, MD, and SD). Specifically, H2O2 content decreased by 30.15%, 28.46%, and 15.29%, and MDA content decreased by 38.05%, 28.69%, and 37.00%.

3.8. Endogenous Protective Enzymes’ Activity of N. tabacum L. After T. harzianum Inoculation During DS Treatment

When plants face biotic and abiotic stresses, endogenous protective enzymes often become active. Therefore, we measured the activities of endogenous protective enzymes in N. tabacum L. under different treatments. The activities of CAT (catalase, Figure 9A), SOD (superoxide dismutase, Figure 9B), POD (peroxidase, Figure 9C), and APX (ascorbate peroxidase, Figure 9D) in the T. harzianum-treated groups (LDTh, MDTh, and SDTh) were higher than in the corresponding drought-treated groups (LD, MD, and SD). Specifically, CAT activity increased by 134.38%, 123.33%, and 157.58%; SOD activity increased by 37.98%, 46.22%, and 18.97%; POD activity increased by 16.67%, 9.90%, and 16.51%; and APX activity increased by 31.73%, 24.92%, and 38.00%.

3.9. Nitrate Reductase Activity of N. tabacum L. After T. harzianum Inoculation During DS Treatment

Nitrate reductase is a rate-limiting enzyme in the nitrogen assimilation of higher plants, directly regulating nitrate reduction and influencing both nitrogen metabolism and photosynthetic carbon metabolism. After measuring nitrate reductase activity, it was found that T. harzianum inoculation (LDTh, MDTh, and SDTh) resulted in higher nitrate reductase activity compared to the respective drought treatments (LD, MD, and SD), with increases of 51.24%, 91.84%, and 378.57%, respectively (Figure S3).

4. Discussion

Trichoderma harzianum, a fungus known for promoting plant growth, offers environmentally friendly advantages over chemical fertilizers and has been widely adopted as a biofertilizer [31]. In this study, a strain of T. harzianum was isolated, demonstrating both plant growth promotion and drought resistance. It was found to colonize plant roots effectively. Under drought conditions, T. harzianum inoculation was evaluated in detail to understand its mechanisms in promoting plant growth and enhancing drought resistance.
To date, many fungi, such as Trichoderma [17,18], arbuscular mycorrhizal fungi [12,15,16], and Penicillium [32,33,34], have been shown to promote plant growth. These fungi enhance seed germination, plant growth, root development, and photosynthesis, ultimately leading to increased crop yields. T. harzianum, as a natural endophytic biocontrol agent, needs to colonize plants to exert its defensive functions [31,35]. For instance, the colonization of T. harzianum in cannabis can promote its growth and development while increasing the content of the active compound cannabidiol (CBD) [36]. After inoculation with Trichoderma sp. T154 in grapes, the fungus can colonize the plant for the long term, located in the xylem, fibers, and parenchyma tissues, thereby providing resistance against Phaeoacremonium minimum infection [37]. Similarly, Trichoderma colonization in Arabidopsis roots enhances tolerance to abiotic stress and resistance to biotic stress [38]. Conversely, a reduction in Trichoderma colonization decreases its ability to enhance plant productivity. It has been shown that Trichoderma fungi improve resistance to both abiotic and biotic stresses, promote nutrient absorption, and stimulate plant growth by enhancing systemic resistance [31,35].
Drought, as one of the most critical environmental factors affecting plant growth, significantly impacts crop yield. This effect is exacerbated by the intensification of the greenhouse effect [39]. Nicotiana tabacum L., an important economic crop, plays a crucial role in the national economy [40]. Additionally, N. tabacum L. serves as a model plant for studying plant physiology and biochemistry, and as a chassis for synthetic biology [20,41,42]. Therefore, researching biocontrol agents that can promote N. tabacum L. growth and resist abiotic stress is urgently needed. This finding indicates that drought stress profoundly affects plant phenotypes, agronomic traits, and root development. However, inoculation with Trichoderma can mitigate drought-induced damage and promote N. tabacum L. growth.
Photosynthesis, the most important process in plant growth and development, is vital for crop yield formation [43]. Abiotic stresses, such as abnormal temperature, water scarcity, and nutrient deficiency, significantly limit photosynthesis, thereby threatening global food security [44,45]. For instance, potassium deficiency severely impacts photosynthesis, leading to reduced growth and crop yield [46]. High temperatures affect photosynthesis by influencing CO2 absorption, photochemical reactions, the turnover of D1 and D2 proteins, and chlorophyll biosynthesis [47]. The results demonstrate that under various levels of drought conditions, inoculating N. tabacum L. with T. harzianum significantly enhances photosynthesis-related parameters and positively impacts the accumulation of chlorophyll a and chlorophyll b.
When plants face extreme environmental conditions such as drought, high temperatures, or high salinity, their growth is severely inhibited, leading to significant oxidative stress and elevated membrane lipid peroxidation indicators [48]. To cope with these stresses, plants secrete osmotic regulatory substances to maintain osmotic balance and mitigate damage [49]. Additionally, plants have evolved a series of antioxidant enzyme systems, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX) [50]. Under varying degrees of drought stress, inoculation with T. harzianum significantly increased the activities of endogenous protective enzymes and the contents of osmotic regulatory substances (soluble sugars, soluble proteins, and proline) in N. tabacum L. Simultaneously, membrane lipid peroxidation indicators (e.g., H2O2 and MDA) showed a significant decrease. These results indicate that inoculation with T. harzianum can reduce the oxidative stress experienced by N. tabacum L. Furthermore, nitrate reductase, an important enzyme in nitrogen assimilation, plays a crucial role in plant growth and development [51]. Recent studies have shown that the activity of nitrate reductase is positively correlated with the concentration of auxin in plant roots, making it a key enzyme in regulating root structure [52]. Our findings demonstrate that inoculation with T. harzianum significantly enhances nitrate reductase activity, resulting in improved root development in N. tabacum L.
The broader application of T. harzianum as a biofertilizer holds significant promise for sustainable agriculture, especially in areas prone to water scarcity. As a biocontrol agent, T. harzianum not only helps in enhancing plant growth but also reduces the reliance on chemical fertilizers, which can have adverse environmental impacts. Its ability to enhance drought tolerance by stimulating the plant’s physiological responses can be a valuable tool in integrated crop management systems aimed at mitigating the effects of climate change. However, there are some limitations to this study that need to be addressed in future research. First, the study was conducted under controlled greenhouse conditions which do not fully replicate the complexity of field environments. Field trials are essential to confirm whether the observed benefits can be consistently achieved under real-world conditions, where factors such as soil type, microbial communities, and weather conditions can influence the outcomes. Furthermore, this study did not include a direct comparison with other biocontrol agents or microbial inoculants, which would provide a more comprehensive understanding of T. harzianum’s relative efficacy in enhancing drought resistance. Future studies should explore the synergy between T. harzianum and other beneficial microbes to determine optimal combinations for enhancing crop performance under stress conditions.
We conclude that the T. harzianum isolated in this study can systematically regulate the growth and development of N. tabacum L. under drought stress, enhancing its adaptability to drought. This study lays the foundation for the development of new, environmentally friendly, and green microbial biofertilizers for crop production.

5. Conclusions

From the rhizosphere of healthy N. tabacum L. plants in a field affected by black shank disease, the plant growth-promoting fungi T. harzianum was successfully isolated. Preliminary studies demonstrated its plant growth-promoting capabilities and its ability to colonize N. tabacum L. roots. Based on these findings, a comprehensive investigation was conducted into the effects of T. harzianum inoculation on plant phenotypes, agronomic traits, photosynthesis, osmotic regulatory substances, lipid peroxidation index, and enzyme activities. The research showed that under varying degrees of drought stress, T. harzianum inoculation significantly improved N. tabacum L. growth and drought resistance. By measuring various growth indicators, it was found that T. harzianum inoculation positively impacted plant growth and resistance to abiotic stress. This study helps researchers better understand plant responses to drought and the mechanisms by which T. harzianum promotes growth and enhances drought resistance, laying the foundation for developing eco-friendly biofertilizers.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms13030543/s1, Figure S1: The plant growth status of N. tabacum L. under different drought conditions (w/o T. harzianum inoculation); Figure S2: The root system status of N. tabacum L. under different drought conditions (w/o T. harzianum inoculation); Figure S3: Nitrate reductase (NR) activity. Figure S4: Original pictures of Figure 2D.

Author Contributions

Conceived and designed the experiments: M.Y. and Q.Y.; performed the experiments: Z.F. and Z.R.; analyzed the data: M.Y., Z.F., A.L. and A.J.; contributed reagents/materials/analysis tools: M.Y., Z.F., Z.R., A.L. and A.J.; wrote the paper: M.Y., Z.F. and A.J. All the authors contributed to editing the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

We appreciate financial support from the National Natural Science Foundation of China (32400218), the Key Technology R&D Program of Henan Province (242102110240, 232102110053), and the Special Support Fund for High-level Talents and skills improvement of Henan Agricultural University (30501474).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Figure 1. Nicotiana tabacum L. with six treatments. (LD, light drought; LDTh, light drought with T. harzianum; MD, moderate drought; MDTh, moderate drought with T. harzianum; SD, severe drought; SDTh, severe drought with T. harzianum).
Figure 1. Nicotiana tabacum L. with six treatments. (LD, light drought; LDTh, light drought with T. harzianum; MD, moderate drought; MDTh, moderate drought with T. harzianum; SD, severe drought; SDTh, severe drought with T. harzianum).
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Figure 2. Trichoderma harzianum isolation from rhizosphere soil of healthy N. tabacum L. (A) Comparison of the healthy and infected N. tabacum L. in field; (B) T. harzianum cultured on potato dextrose agar media; (C) promoting-growth effect after T. harzianum inoculation (CK: control; Th: T. harzianum); (D) colonization of T. harzianum in N. tabacum L. roots.
Figure 2. Trichoderma harzianum isolation from rhizosphere soil of healthy N. tabacum L. (A) Comparison of the healthy and infected N. tabacum L. in field; (B) T. harzianum cultured on potato dextrose agar media; (C) promoting-growth effect after T. harzianum inoculation (CK: control; Th: T. harzianum); (D) colonization of T. harzianum in N. tabacum L. roots.
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Figure 3. Enhancement of N. tabacum L.’s agronomic traits after T. harzianum inoculation under light drought (LD), moderate drought (MD), and severe drought (SD). (A) Height; (B) stem grith; (C) maximum leaf width; (D) maximum leaf length; (E) number of effective leaves.
Figure 3. Enhancement of N. tabacum L.’s agronomic traits after T. harzianum inoculation under light drought (LD), moderate drought (MD), and severe drought (SD). (A) Height; (B) stem grith; (C) maximum leaf width; (D) maximum leaf length; (E) number of effective leaves.
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Figure 4. Enhancement of N. tabacum L.’s root growth and development after T. harzianum inoculation under light drought (LD), moderate drought (MD), and severe drought (SD). (A) Total root length; (B) root projection area; (C) root surface area; (D) root volume; (E) root diameter; (F) root tip member; (G) branch number; (H) number of connection; (I) root activity.
Figure 4. Enhancement of N. tabacum L.’s root growth and development after T. harzianum inoculation under light drought (LD), moderate drought (MD), and severe drought (SD). (A) Total root length; (B) root projection area; (C) root surface area; (D) root volume; (E) root diameter; (F) root tip member; (G) branch number; (H) number of connection; (I) root activity.
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Figure 5. Effect of drought and T. harzianum inoculation on photosynthetic parameters. (A) Maximum net photosynthetic rate; (B) stomatal conductance; (C) intercellular CO2 concentration; (D) transpiration rate; (E) relative water content.
Figure 5. Effect of drought and T. harzianum inoculation on photosynthetic parameters. (A) Maximum net photosynthetic rate; (B) stomatal conductance; (C) intercellular CO2 concentration; (D) transpiration rate; (E) relative water content.
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Figure 6. Effect of drought and T. harzianum inoculation on the photosynthetic pigments content of in N. tabacum L. leaves. (A) Chlorophyll a; (B) chlorophyll b; (C) ratio of chlorophyll a to b.
Figure 6. Effect of drought and T. harzianum inoculation on the photosynthetic pigments content of in N. tabacum L. leaves. (A) Chlorophyll a; (B) chlorophyll b; (C) ratio of chlorophyll a to b.
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Figure 7. Effect of drought and T. harzianum inoculation on the content of osmoprotectants in N. tabacum L. leaves. (A) Soluble sugar; (B) soluble protein; (C) proline.
Figure 7. Effect of drought and T. harzianum inoculation on the content of osmoprotectants in N. tabacum L. leaves. (A) Soluble sugar; (B) soluble protein; (C) proline.
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Figure 8. Effect of drought and T. harzianum inoculation on membrane lipid peroxidation index in N. tabacum L. leaves. (A) H2O2; (B) MDA.
Figure 8. Effect of drought and T. harzianum inoculation on membrane lipid peroxidation index in N. tabacum L. leaves. (A) H2O2; (B) MDA.
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Figure 9. Effect of drought and T. harzianum inoculation on endogenous protective enzymes in N. tabacum L. leaves. (A) CAT; (B) SOD; (C) POD; (D) APX.
Figure 9. Effect of drought and T. harzianum inoculation on endogenous protective enzymes in N. tabacum L. leaves. (A) CAT; (B) SOD; (C) POD; (D) APX.
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Yin, Q.; Feng, Z.; Ren, Z.; Li, A.; Jaisi, A.; Yang, M. Rhizosphere Growth-Promoting Fungi of Healthy Nicotiana tabacum L.: A Systematic Approach to Boosting Plant Growth and Drought Resistance. Microorganisms 2025, 13, 543. https://doi.org/10.3390/microorganisms13030543

AMA Style

Yin Q, Feng Z, Ren Z, Li A, Jaisi A, Yang M. Rhizosphere Growth-Promoting Fungi of Healthy Nicotiana tabacum L.: A Systematic Approach to Boosting Plant Growth and Drought Resistance. Microorganisms. 2025; 13(3):543. https://doi.org/10.3390/microorganisms13030543

Chicago/Turabian Style

Yin, Quanyu, Zhao Feng, Zhichao Ren, Ao Li, Amit Jaisi, and Mengquan Yang. 2025. "Rhizosphere Growth-Promoting Fungi of Healthy Nicotiana tabacum L.: A Systematic Approach to Boosting Plant Growth and Drought Resistance" Microorganisms 13, no. 3: 543. https://doi.org/10.3390/microorganisms13030543

APA Style

Yin, Q., Feng, Z., Ren, Z., Li, A., Jaisi, A., & Yang, M. (2025). Rhizosphere Growth-Promoting Fungi of Healthy Nicotiana tabacum L.: A Systematic Approach to Boosting Plant Growth and Drought Resistance. Microorganisms, 13(3), 543. https://doi.org/10.3390/microorganisms13030543

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