Endophytic extract Zhinengcong alleviates salt stress-induced reproductive defects in Solanum lycopersicum

Abstract

Salt stress severely impairs reproductive development in tomato plants, leading to reduced pollen viability, compromised pollen germination, abnormal pollen tube growth, and ultimately substantial yield losses. Zhinengcong (ZNC), a crude extract derived from the endophytic fungus Paecilomyces variotii isolated from wild sea buckthorn, has previously been reported to enhance plant resistance to abiotic stresses. Here, we demonstrate that ZNC effectively alleviates salt stress induced reproductive defects in tomato, restoring pollen viability and pollen tube growth and attenuating developmental abnormalities in reproductive tissues. Mechanistically, ZNC treatment downregulates the expression of key reactive oxygen species (ROS)-producing genes, including RBOHs, while upregulating antioxidant-related genes and enhancing the activities of corresponding antioxidant enzymes. These coordinated responses suppress excessive ROS accumulation in anthers, pollen grains, and pollen tubes under salt stress. Together, our findings reveal a protective role of the endophytic fungal extract ZNC in mitigating salt-induced reproductive inhibition and suggest its potential application for improving tomato reproduction in saline-alkaline soils.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08367-3.

Introduction

The continuous expansion of salinized soils has become an increasingly serious challenge for agriculture, particularly in northern China [1–3]. Severe soil salinization markedly impairs seed germination, vegetative growth, and crop yield [4]. Among different developmental stages, reproductive development is especially sensitive to salt stress and plays a decisive role in yield formation in most flowering crops. A previous study has shown that salt stress negatively affects both pollen and stigma, with pollen exhibiting significantly higher sensitivity than stigma [5]. Salt stress disrupts pollen development, anther dehiscence, pollen fertility, and pollen tube growth, frequently leading to male sterility and consequent yield reduction in crops [5–8]. Therefore, identifying effective strategies to alleviate salt stress is of great importance for the sustainable utilization of salinized agricultural regions.

Tomato (Solanum lycopersicum L.) is one of the most economically important vegetable crops worldwide. Despite its extensive cultivation in saline-alkaline regions, most modern tomato cultivars remain sensitive to moderate soil salinity [9]. Salt stress adversely affects tomato reproductive development by suppressing floral primordia formation [10], inducing early flowering and flower abscission, and reducing the production of viable pollen [11, 12]. At the same time, moderate salinity can improve certain fruit quality traits by increasing the accumulation of flavor-related compounds [13, 14]. These contrasting effects highlight the need for approaches that mitigate the negative impacts of salt stress on reproduction without compromising fruit quality.

Zhinengcong (ZNC) is a bioactive extract derived from the plant endophytic fungus Paecilomyces variotii and is currently used as a commercial agricultural input incorporated into fertilizers. Although the precise chemical composition of ZNC has not yet been characterized, previous studies have shown that it can significantly promote plant growth, enhance resistance to biotic stresses such as pathogens, viruses, and Botrytis cinerea [15, 16], improve thermotolerance, and promote tomato pollen tube elongation under normal growth conditions [17]. However, whether ZNC can enhance plant tolerance to salt stress, particularly during reproductive development, remains unclear.

Abiotic stress strongly disrupts protein, lipid, and carbohydrate metabolism and alters reactive oxygen species (ROS) homeostasis in plants [5]. ROS, including superoxide anion (O₂·⁻), hydroxyl radical (·OH), and hydrogen peroxide (H₂O₂), function not only as cytotoxic molecules but also as essential signaling components in plant development and stress responses [18–20]. During anther development, maintenance of a low ROS environment during the pre-meiotic stage is crucial for proper differentiation of anther primordium cells [21, 22]. In contrast, stage-specific ROS accumulation in tapetal cells during late meiosis triggers programmed cell death (PCD), a process required for normal pollen maturation [23, 24]. Under abiotic stress conditions, excessive ROS accumulation in anthers can induce premature tapetal cell death, thereby disrupting pollen development and leading to pollen sterility [7]. ROS also play critical roles during pollen germination and pollen tube growth [25–28]. Elimination of ROS using ROS scavengers significantly inhibits pollen tube growth in kiwi [25], whereas high-temperature stress induces excessive ROS accumulation in tomato pollen tubes, inhibiting elongation and potentially causing pollen tube rupture [17]. Together, these findings indicate that precise regulation of ROS is required for normal pollen development and pollen tube growth.

In this study, we show that ZNC alleviates salt stress-induced reproductive defects in tomato and improves yield under salt stress. Our findings reveal a previously unrecognized role of ZNC in regulating reproductive development under salt stress and suggest its potential application for improving tomato production in salinized soil.

Results

Salt stress reduces pollen viability and inhibits pollen germination and growth

Consistent with previous report [29], salt stress significantly inhibited vegetative growth (Supplementary Fig. S1A-E). We collected pollen grains from plants subjected to different salt treatments and germinated them in vitro. The germination rate decreased markedly from 85.79% in the control to 68.86%, 39.71%, and 13.66% under 50 mM, 100 mM, and 150 mM NaCl treatments, respectively (Fig. 1A, B). Pollen tube length was also significantly shorter under salt stress (Fig. 1A, C). We assessed pollen viability using fluorescein diacetate (FDA) and propidium iodide (PI) dual staining. Pollen viability declined sharply under salt stress, decreasing from 85.28% in control plants to 53.84% and 34.46% following 100 mM and 150 mM NaCl treatments, respectively. (Fig. 1A, D). At the same time, we observed a clear increase in morphologically abnormal pollen grains with rising salt concentrations (Fig. 1A, E). In addition, the growth of 100 mM and 150 mM salt treated pollen on normal stigmas were severely inhibited with the increasing of salt concentration (Fig. 1F-I). Salt stress at 150 mM NaCl inhibited normal pollen tube growth on stigmas (Supplementary Fig. S2A-C). Altogether, our results demonstrate that salt stress severely impairs pollen viability, germination, and pollen tube growth in tomato.

Fig. 1.

Salt stress reduces pollen viability and pollen tube growth. A Pollen germination, pollen tube growth, and pollen viability in tomato plants subjected to salt stress. Pollen viability was assessed by fluorescein diacetate (FDA) and propidium iodide (PI) dual staining, where green fluorescence indicates viable pollen and red fluorescence indicates nonviable pollen. Scale bars, 200 μm (upper panel) and 100 μm (lower panel). B Quantification of pollen germination rates shown in (A). C Quantification of pollen tube lengths shown in (A). D Quantification of pollen viability rates shown in A. E Quantification of pollen malformation rates shown in (A). F Growth of salt stress-treated pollen in normal pistils. Scale bar, 500 μm. G Quantification of pollen tube number shown in (F). H Quantification of pollen tube lengths shown in (F). Box plots: center line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points. Asterisk on the top of data box indicates significant difference (two-tailed t-test, *p < 0.05, **p < 0.01) compared with the data on the far left of their own group, while asterisk above the bracket represents comparison between the two data bars indicated. All experiments were performed with three biological replicates, and similar results were obtained. The same applies to subsequent figures

ZNC alleviates the inhibitory effects of salt stress on pollen viability and germination

To investigate whether ZNC alleviates the inhibitory effects of salt stress on tomato pollen, we exogenously applied ZNC at concentrations ranging from 10 to 30 ng/mL to tomato plants [17] exposed to 100 mM NaCl. ZNC treatment markedly restored pollen performance under salt stress. Among the tested concentrations, 20 ng/mL ZNC showed the strongest mitigative effect, increasing the pollen germination rate from 36.83% under salt stress to a maximum of 78.10% and extending pollen tube length from 125.80 μm to 179.40 μm (Fig. 2A-C). We next assessed pollen viability using FDA/PI staining. ZNC treatment significantly increased pollen viability under salt stress, from 55.91% in salt-stressed plants to 68.50–96.20% in ZNC-treated groups (Fig. 2A, D). To evaluate pollen performance in vivo, we pollinated normal stigmas with pollen from salt-stressed plants treated with ZNC. We observed a significant increase in the number of penetrating pollen tubes in all ZNC-treated groups (10–30 ng/mL), and pollen tube lengths were significantly greater than those observed under 100 mM NaCl stress alone (Fig. 2E-G). Scanning electron microscopy revealed that 52.00% of pollen grains displayed abnormal morphology following salt stress treatment, whereas application of 20 ng/mL ZNC reduced the proportion of abnormal pollen grains to 13.80% (Fig. 2H, I). In addition to its effects on reproductive traits, we found that ZNC application also promoted the growth of tomato leaves and roots under salt stress (Supplementary Fig. S3A-D). Taken together, these results demonstrate that ZNC effectively alleviates salt stress-induced inhibition of pollen viability, germination, and pollen tube growth in tomato.

Fig. 2.

ZNC alleviates salt stress-induced impairment of pollen viability and pollen tube growth. A Pollen germination, pollen tube growth, and pollen viability in tomato plants treated with different concentrations of ZNC and exposed to 100 mM NaCl for 4 weeks. ZNC was sprayed every 3 days. Scale bars, 200 μm (upper panel) and 100 μm (lower panel). B Quantification of pollen germination rates shown in (A). C Quantification of pollen tube lengths shown in A. D Quantification of pollen viability rates shown in (A). E Germination and growth of pollen pretreated with different concentrations of ZNC on stigmas grown under normal conditions for 4 h. Scale bar, 500 μm. F Quantification of pollen tube number shown in (E). G Quantification of pollen tube lengths shown in (E). H, I Scanning electron microscopy (SEM) images of pollen morphology and quantification of pollen malformation rates in tomato plants pretreated with 0 or 20 ng/mL ZNC under normal growth conditions or 100 mM NaCl stress. Scale bars, 100 μm (upper panel) and 10 μm (lower panel)

ZNC suppresses salt stress-induced excessive ROS accumulation in pollen

Abiotic stress disrupts cellular metabolic balance and causes excessive accumulation of ROS [18]. To investigate whether ZNC alleviates salt stress-induced inhibition of pollen development by regulating ROS levels, we stained pollen grains from different treatments with H₂DCFDA to detect intracellular ROS. We observed a marked increase in ROS levels in pollen grains under salt stress, whereas ZNC treatment significantly reduced ROS accumulation under the same salt stress conditions (Fig. 3A). We obtained similar results from nitroblue tetrazolium (NBT) staining of pollen tubes. Salt stress caused pronounced ROS accumulation at pollen tube tips, while ZNC treatment effectively alleviated salt-induced ROS accumulation (Fig. 3B, C). These results indicate that the alleviative effects of ZNC on salt stress-induced inhibition may involve the reduction of excessive ROS accumulation.

Fig. 3.

ZNC enhances pollen antioxidant capacity and reduces salt stress-induced reactive oxygen species accumulation. A H₂DCFDA-stained pollen grains showing intracellular ROS accumulation under different treatments. ROS levels in pollen grains without salt stress and ZNC treatment were set to 1 for comparative analysis. Number of observed pollen grains, n = 50. Scale bar, 200 μm. B, C NBT-stained pollen tubes showing ROS accumulation under different treatments. ROS levels in pollen tubes without salt stress and ZNC treatment were set to 1 for comparative analysis. Scale bar, 50 μm. D Expression levels of NADPH oxidase genes RBOHB and RBOHE in tomato pollen under different treatments. E Expression levels of antioxidant-related genes Fe-SOD, DHAR, and CAT1 in tomato pollen under different treatments. F Enzymatic activities of SOD, POD, and CAT in tomato pollen under different treatments. G Expression levels of P5CS1 in tomato pollen under different treatments. Expression levels in pollen grains without salt stress and ZNC treatment were set to 1 for comparative analysis, and normalized to the housekeeping gene ACTIN (GenBank accession number AB199316). Data bar (D, E, F, G): average ± SD (n = 3 independent biological replicates per treatment)

NADPH oxidases (RBOHs) play key roles in ROS production in plants. Among them, RBOHB and RBOHE are highly expressed in mature anthers and pollen and are known to regulate pollen development and pollen tube growth by modulating ROS levels [30, 31]. To explore the correlation of ZNC and the reduced ROS accumulation under salt stress, we first examined the expression of RBOHB and RBOHE in anthers. We found that salt stress treatment with 100 mM NaCl significantly induced the expression of both genes, whereas treatment with 20 ng/mL ZNC markedly suppressed their expression under salt stress (Fig. 3D). We next analyzed the expression of genes associated with the antioxidant system. ZNC treatment slightly upregulated the expression of Fe-SOD, DHAR, and CAT1 under normal growth conditions and significantly enhanced their expression in anthers under salt stress (Fig. 3E). Consistently, enzyme activity assays showed that ZNC treatment significantly increased the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in anthers under salt stress (Fig. 3F). Proline accumulation in pollen grains as a potential target for improving yield stability under salt stress [32]. Our results indicate that ZNC treatment upregulates the expression of P5CS1, a key gene in the proline synthesis pathway, under salt stress conditions (Fig. 3G). Together, these results suggest that ZNC induced-ROS scavenging under salt stress may mediated by both suppressing ROS production through downregulation of RBOHB and RBOHE and enhancing ROS scavenging capacity via activation of antioxidant gene expression and enzyme activities, thereby alleviating ROS-associated damage during pollen development under salt stress.

ZNC improves fruit and seed number in tomato under salt stress

Because ZNC significantly improved pollen viability under salt stress, we next investigated whether ZNC treatment also affects flowering, fruit set, and yield in tomato plants exposed to salinity. We found that ZNC treatment increased the number of flowers produced under salt stress (Fig. 4A, B) and significantly reduced flower drop, which decreased to 19.74% under salt stress conditions (Fig. 4A, C). As a consequence, ZNC-treated plants produced more fruits and seeds, resulting in an almost twofold increase in yield compared with salt-stressed plants without ZNC treatment (Fig. 4D-G). Salt stress increased fruit sugar accumulation, consistent with previous observations. ZNC treatment slightly attenuated this effect, leading to a modest reduction in sugar content under salt stress (Fig. 4H). Overall, these results indicate that ZNC application effectively improves fruit set, seed production, and yield in tomato under salt stress.

Fig. 4.

ZNC improves fruit and seed production in tomato under salt stress. A Hydroponically grown tomato plants treated with 0 or 20 ng/mL ZNC (sprayed every 3 days) under 100 mM NaCl for 5 weeks. Scale bars, 15 cm (left panel) and 0.5 cm (right panel). B Quantification of flower number shown in (A). C Quantification of flower drop rates shown in (A). D Hydroponically grown tomato plants treated with 0 or 20 ng/mL ZNC (sprayed every 3 days) under 100 mM NaCl until fruit and seed set. Scale bars, 15 cm (upper panel) and 1 cm (lower panel). E Quantification of fruit number shown in (D). F Quantification of fruit yield shown in (D). G Quantification of seed number per fruit shown in (D). H Quantification of sugar content shown in (D)

Materials and methods

Plant materials and treatments

Tomato cultivar ‘Micro-Tom’ seeds were obtained from Professor Qinghua Shi (Shandong Agricultural University). Plants were grown in soil under controlled conditions at 25/20°C (day/night) with a 16/8 h photoperiod for 3 weeks, and then transferrde to a hydroponic system. During the pretreatment phase, plants were sprayed twice weekly at 3-day intervals for 1 week. After pretreatment, plants were transferred to salt stress conditions ranging from mild to severe (50, 100, and 150 mM NaCl) [33]while maintaining the same spraying frequency of once every 3 days. Each biological replicate consisted of at least 10 individual plants.

Pollen germination and viability assays

Pollen viability, germination, and morphology were assessed from the same biological samples. Pollen grains from different treatment groups were collected and cultured on pollen germination medium (PGM) containing 24% PEG4000, 1.6 mM H₃BO₃, 2% sucrose, 20 mM HEPES (pH 6.0), 30 mM Ca(NO₃)₂, 100 µM MgSO₄, and 5 mM KCl. Pollen cultures were incubated at 25 ± 1 °C for 2 h, after which pollen germination rates and pollen tube lengths were measured.

Pollen viability was assessed using a fluorescence double-staining method. Pollen grains were incubated in pollen viability solution (PVS) containing 0.001% fluorescein diacetate (FDA), 20 µM propidium iodide (PI), 290 mM sucrose, 1.27 mM Ca(NO₃)₂, 0.16 mM H₃BO₃, and 1 mM KNO₃ for 10 min in the dark before observation.

To analyze pollen tube growth in vivo, pollen from salt-stressed or ZNC treated plants was manually pollinated onto untreated stigmas. After 6 h, stigmas were fixed in Carnoy’s solution (methanol: glacial acetic acid = 3:1) and stained with 0.1% aniline blue, following a previously reported method [34]. Pollen tube growth was observed using a fluorescence microscope (Nikon Eclipse Ni equipped with a DS-Ri 2 camera).

SEM observation

Pollen samples from different treatments were mounted onto metal specimen stubs using conductive adhesive tape to ensure uniform distribution. Samples were then sputter-coated with a thin layer of gold to improve conductivity. The coated specimens were subsequently examined using a scanning electron microscope under high-vacuum conditions. All scanning electron microscopy observations were conducted at Shandong Agricultural University (Shandong, China) using a JSM-6610LV instrument. Systematic observation and documentation of pollen surface morphology were performed at magnifications of 220× and 2500×.

ROS staining assays

Intracellular ROS levels in pollen grains were detected using the fluorescent probe H₂DCFDA and NBT. For H₂DCFDA staining, pollen grains were incubated in PVS containing 20 µM H₂DCFDA for 10 min in the dark, washed three times with PVS, and then imaged. To visualize ROS accumulation in pollen tubes, pollen grains were cultured in PGM for 40 min and stained with 2 mg/mL NBT for 8 min, as previously described [35]. Images were captured using a Nikon Eclipse Ni microscope equipped with a DS-Ri 2 camera. ROS signal intensity was quantified using ImageJ.

Antioxidant enzyme activity assays

Anthers of newly open flowers (0.5 g) from salt-stressed or ZNC-treated plants (8 plants per treatment.) were flash-frozen in liquid nitrogen, homogenized, and resuspended in 2 mL of phosphate buffer (0.05 M, pH 7.8). After centrifugation at 12,000 × g for 20 min at 4 °C, the supernatant was collected for enzyme activity measurements. Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were determined using established methods. SOD activity was measured using the NBT photoreduction method [36], with one unit (U) defined as the amount of enzyme required to inhibit 50% of NBT reduction. POD activity was assayed by monitoring guaiacol oxidation at 470 nm [37]. CAT activity was quantified by measuring H₂O₂ consumption at 240 nm [37].

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from anthers subjected to different treatments using the DP420 kit (Tiangen) and subsequently reverse-transcribed into cDNA. Quantitative real-time PCR was performed using ChamQ SYBR qPCR Master Mix (Q711-03, Vazyme) on a qTOWER3 system (Analytik Jena, Germany). The thermal cycling protocol was as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 10 s. A dissociation step from 65 °C to 95 °C with increments of 0.5 °C for 5 s per step was performed to confirm amplification specificity. Data are presented relative to the untreated control condition (without salt stress and ZNC treatment) and normalized to the housekeeping gene ACTIN (GenBank accession number AB199316), with three biological replicates per sample. Primer sequences used for qRT-PCR are listed in Table S1.

Measurement of root length and leaf area

Root length was measured manually using a standard ruler. Leaf area was determined using ImageJ software based on leaves collected from the mid-height region of each plant.

Sugar content determination

Sugar contents of fully ripe tomato fruit were determined by using a handheld digital refractometer (measuring range: 0.0%-35.0% Brix, accuracy: ± 0.2% Brix), Measurements were repeated six times per fruit, and the average value was calculated.

Statistical analysis

All data are presented as bar or box plots with individual data points shown and were generated using GraphPad Prism. Statistical significance was indicated by asterisks above bars relative to the leftmost control group (two-tailed t-test, *p < 0.05, **p < 0.01), while asterisk above the bracket represents comparison between the two data bars indicated. The symbol n.s. denotes no significant difference (p ≥ 0.05). Bracketed asterisks indicate direct pairwise comparisons between groups. All experiments were performed with three biological replicates.

Discussion

Tomato yield largely depends on successful fruit set, a process that is highly sensitive to reproductive performance. Abiotic stresses, particularly salt stress, severely impair pollen development, pollen germination, and pollen tube growth, ultimately leading to substantial reductions in crop yield and quality [5–8].

At the reproductive level, salt stress induces excessive ROS accumulation in anther tissues, triggering premature PCD of tapetal cells. Early degradation of the tapetum compromises nutrient supply and sporopollenin deposition required for pollen development, ultimately leading to pollen abortion and impaired fertilization [23, 24]. In addition, elevated ROS levels cause oxidative damage to proteins, DNA, and lipids [38]. Consistently, application of the antioxidant ascorbic acid or the NADPH oxidase inhibitor DPI has been shown to rescue high temperature-induced defects in pollen tube growth and integrity in tomato, highlighting the importance of ROS removal for maintaining pollen viability and pollen tube growth under abiotic stress conditions [39]. Our results demonstrate that ZNC treatment contributes to the effective reduction of salt stress-induced ROS accumulation in pollen grains, pollen tube tips, and anthers, thereby supporting normal pollen development, reducing pollen deformity, and improving pollen viability (Fig. 3).

Besides ZNC, many other biostimulants, including algal extracts, protein hydrolysates, humic and fulvic acids, have also been reported to enhance plant abiotic stress tolerance, with many already formulated as commercial products. Although their bioactive components and molecular mechanisms may vary, the scavenging of stress-induced ROS is a common response for most of these biostimulants [40–43].

ZNC exhibits pronounced plant growth regulatory activity at very low concentrations (1-100 ng/mL) and low application cost. It has previously been shown to promote root development in Arabidopsis and to confer broad-spectrum resistance against bacterial, viral, and oomycete pathogens in multiple plant species [15, 44]. Here, we demonstrate that ZNC also alleviates salt stress-induced reproductive defects in tomato, supporting its potential as a viable strategy for enhancing crop productivity in saline-alkaline agricultural systems. Although ZNC may broadly enhance ROS homeostasis under various stress conditions, whether it acts through salt stress-specific pathways remains to be further investigated.

In this study, ZNC significantly mitigate salt stress in reproductive process, which makes ZNC a potential stimulator in tomato production in salinized soil. However, our experiments were designed in very small scales with a widely used experimental tomato cultivar for laboratory and growth chamber studies. It is essential to repeat the experiments in field with larger amount of samples and commercial cultivars, and test more agronomical traits to prove its ability in improving salt tolerance.

While studies on salt stress have mainly focused on vegetative growth defects, its impact on reproductive processes remains less understood, despite observed effects such as reduced fruit set and loss of pollen viability. In our study, we found that ZNC may exert its mitigating effects through ROS scavenging, which is a common strategy of ZNC and many other exogenous biostimulants in alleviating various abiotic stresses. The molecular and physiological mechanisms that are specifically triggered by salt stress during reproduction remain largely unknown and must be further investigated.

Authors’ contributions

The authors confirm their contribution to the paper as follows: study conception and design: QD, YC, and RL; data collection: RC, RL, XC, and HD; analysis and interpretation of results: RC and RL; manuscript drafting: YC, RC, XC, XZ, BK, and LD. All authors reviewed the results and approved the final version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32573038 to Y.C., U24A20418 to Q.D.), the Shandong Natural Science Foundation (ZR2025MS402 to Y.C.), the Key R&D Program of Shandong Province, China (2024LZGC036 to Y.C. and R.L.), and Shandong Pengbo Biotech Limited Company.

Data availability

The original contributions presented in the study are included in the article and Supplementary materials. Further inquiries can be directed to the corresponding authors.

Declarations

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Competing interests

The authors declare no competing interests.