Introduction

Insect infestation significantly impacts plant growth, biomass accumulation, and physiological characteristics, often inducing plant defense responses and altering endogenous substances1. Numerous studies have demonstrated that pest infestations lead to changes in various growth indices of host plants. For instance, Chen et al. observed that plant height and leaf area of the hybrid rice genotype Shanyou 63 increased after infestation by Nilaparvata lugens at densities of 10 and 20 heads per plant2. Similarly, infestation by Frankliniella occidentalis resulted in decreased levels of soluble sugar, soluble protein, free amino acid, and chlorophyll in Phaseolus vulgaris leaves, with the extent of reduction correlating with insect population density3. In tomato (Solanum lycopersicum), the concentrations of soluble sugar, soluble protein, and free amino acid were positively correlated with density and duration of Etranychus urticae infestation4. Conversely, chili peppers (Capsicum annuum) infested by Bemisia tabaci exhibited higher chlorophyll and soluble sugar content compared to non-infested plants5.

Bradysia impatiens, commonly known as the fungus gnat, is a major pest found in mushroom houses, greenhouses, flower pots, nurseries, and other environments6. The larvae of this pest have a broad dietary range, feeding on the rhizomes of various plants, including Chinese chive (A. tuberosum), onion (A. fistulosum), garlic (A. sativum), and strawberry (Fragaria× ananassa). Carbon dioxide (CO2) is a critical raw material for photosynthesis in green plants, enabling them to convert CO2 into energy-rich compounds necessary for growth7. The concentration of CO2 in the environment directly influences on plant growth and crop yield8. Elevated CO2 (eCO2) concentrations have been shown to reduce stomatal conductance in plant leaves, thereby weakening transpiration rates9,10,11,12, and improving water use efficiency13. Consequently, it is widely accepted that eCO2 enhances photosynthetic efficiency, accelerates plant growth, increases the carbon-to-nitrogen ratio, and reduces nitrogen content in plants14,15.

Recent research has also explored the interaction between eCO2 and plant-pest dynamics. Elevated CO2 levels can alter plant physiology and biochemistry, potentially affecting the susceptibility of plants to insect pests. For example, some studies suggest that eCO2 may reduce the nutritional quality of plants for herbivores by decreasing nitrogen content, while others indicate that it may enhance plant resistance to certain pests by increasing the production of secondary metabolites16,17. However, the specific effects of eCO2 on plant-pest interactions, particularly in the context of B. impatiens infestation, remain poorly understood.

Chinese chive is widely cultivated in China due to its unique flavor and nutritional benefits. Despite its economic importance, there is a significant gap in our understanding of how B. impatiens infestation affects Chinese chive under eCO2 conditions. This study is based on the hypothesis that Chinese chive plants exposed to different levels of eCO2 will exhibit varying responses to B. impatiens infestation. The research aims to evaluate the physiological and growth responses of Chinese chive to varying levels of fungus gnat infestation under controlled CO2 conditions in growth chambers, thereby providing insights into the complex interactions between eCO2, plant physiology, and pest densities.

Materials and methods

Plant materials and growth conditions

The experiment was conducted at the Laboratory of Insect Ecology, College of Plant Protection, Gansu Agricultural University, Lanzhou, China. The Chinese chive cultivar “Pingjiu 2” used in the study, was procured from Lanzhou Shengshi Agricultural Seed Company, China. Seeds were sown in pots (10 cm diameter× 10 cm height) containing 1.3 kg of loamy soil. Subsequently, the pots were transferred into two CO2 growth chambers with distinct CO2 concentrations: one set to ambient CO2 (aCO2 = 400 µL/L) and the other to elevated CO2 (eCO2 = 800 µL/L). The growth chamber maintained a controlled environment at 25 ± 1 ℃, with a relative humidity of 50 ± 10% and a 16: 8 h light: dark photoperiod. The plant was cultivated for three months prior to experiment use.

Larvae of B. impatiens were collected from Chinese chive plants in cultivation greenhouses located in Tianshui, China (34°45′22′′ N, 105°7′2′′ E). The larvae were reared on Chinese chive bulbs in Petri dishes under controlled conditions of 25 ± 1 ℃, 50 ± 10%relative humidity with 16: 8 h light: dark photoperiod.

Experimental design

The experiment followed a completely randomized design with three fungus gnat density treatments, each replicated six times. The study incorporated two CO2 concentration levels (aCO2 = 400 µL/L, eCO2 = 800 µL/L), three fungus gnat density levels (0, 10, and 30 larvae per plant). A total of 2 × 3 × 6 = 36 experimental pots were used, with each experimental unit consisting of six pots, each containing one plant. Second-instar larvae of B. impatiens were introduced to the Chinese chive plants 15 days after sowing. Throughout the experiment, the plants were watered adequately to maintain optimal soil moisture levels.

Measurement of growth parameters in Chinese chives

One month after fungus gnat infestation, growth parameters of Chinese chive plants were evaluated, including leaf width (LW), leaf thickness (LT), stem diameter (SD), and plant height (PH). Vernier calipers were used to measure LW, LT and SD. For LW, the widest part of the leaf blade was measured perpendicular to the midrib. LT was measured at the thickest point of the leaf by gently closing the calipers around the leaf to avoid tissue damaging. SD was measured at the midpoint of the stem, ensuring the calipers were perpendicular to the stem axis. Multiple measurements were taken for each parameter to ensure accuracy, and the average value was recorded. PH was measured using a ruler, from the base of the plant (soil level) to the tip of the longest leaf or stem. Care was taken to maintain the plant in an upright position during measurement.

Analysis of nutritional composition in Chinese chives

For each treatment, 0.1 g of Chinese chive samples were collected, immediately frozen in liquid nitrogen, and processed in six replicates. These samples were analyzed to determine the contents of soluble sugar (SS), soluble protein (SP), free amino acid (FAA), and free fatty acid (FFA) using standard biochemical assays and commercially avaiable kits (purchased from Beijing Solebao Technology Co., Ltd.)18. SS were quantified using the anthrone-sulfuric acid method, which involves the reaction of sugars with anthrone reagent under acidic conditions to produce a blue-green color, measurable spectrophotometrically at 620 nm. SP were quantified using the Bradford assay, which relies on the binding of Coomassie Brilliant Blue dye to proteins, resulting in a color change measurable at 595 nm. FAA were quantified using the ninhydrin method, where ninhydrin reacts with amino acids to produce a purple color, measured spectrophotometrically at 570 nm. FFA were quantified using a colorimetric assay based on the formation of a copper-soap complex, which reacts with a chromogenic agent, producing a measurable color change, and absorbance was measured at 440 nm. All measurements were performed following the manufacturer’s instructions, and appropriate controls were included to ensure accuracy and reproducibility.

Determination of photosynthetic pigments in Chinese chives

Chlorophyll a (Ch-a), chlorophyll b (Ch-b), total chlorophyll (Ch), and carotenoid (Ca) content were measured using a portable chlorophyll meter (CCM-200, Opti-Sciences, Tyngsboro, MA, USA) following the methodology described by Neto et al.19. Healthy, fully expanded leaves were selected from each plant. The leaves were cleaned to move dust or debris, as these could interfere with light transmission. The CCM-200 was calibrated according to the manufacturer’s instructions using a calibration standard. The leaf was placed in the meter’s sample holder, ensuring full contact between the leaf and the sensor. The meter emits light at specific wavelengths and measures the amount of light transmitted through the leaf. Ch-a absorbs light most strongly in the red region (around 660 nm), while Ch-b absorbs more in the blue region (around 640 nm). Carotenoids absorb strongly in the blue region (around 470 nm).

Statistical analysis

Statistical analysis was conducted using SPSS Statistics software (version 19.0, SPSS, Chicago, IL, USA). The data were subjected to analysis of variance (ANOVA) to determine the significance of differences among treatment means. For parameters where significant differences were detected (P < 0.05), Duncan’s multiple range test was applied as a post-hoc analysis to separate and compare treatment means. The results are presented as means ± standard error (SE) to provide a measure of variability within the data.

Results

Effects of eCO2 concentration and B. impatiens infestation on growth parameters in Chinese chives

The growth indices of Chinese chive, including leaf width (LW), leaf thickness (LT), plant height (PH), and stem diameter (ST), showed significant changes after infestation by B. impatiens under eCO2 conditions (Fig. 1). Under the same population density of B. impatiens, LW and ST increased with rising CO2 concentrations. The difference in LW between aCO2 and eCO2 was highly significant (P < 0.01; Fig. 1A), while the difference in SD was significant (P < 0.05; Fig. 1D). In contrast, PH decreased with increasing CO2 concentration, showing a significant difference between aCO2 and eCO2 at 30 larvae/plant (P < 0.05; Fig. 1C). No significant differences were observed in LT between aCO2 and eCO2 (P > 0.05; Fig. 1B). At the same CO2 concentration, the highest values for LW, LT, and SD were observed at 10 larvae/plant, while the lowest values occurred at 30 larvae/plant.

Fig. 1
figure 1

Effects of eCO2 concentration and B. impatiens infestation on growth parameters in Chinese chives. (A) Leaf width; (B) Leaf thickness; (C) Plant height; (D) Stem diameter. Asterisks of *, ** and ns respectively represent P < 0.05, P < 0.01, and P > 0.05 between two CO2 concentration. Lowercase letters of a, b and c denote significant differences (P < 0.05) among three infestation densities at 400 µL/L. Capital letters of A, B and C indicate significant differences (P < 0.05) among three infestation densities at 800 µL/L.

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Effects of eCO 2 concentration and B. impatiens infestation on nutritional composition in Chinese chives

The contents of soluble sugar (SS), soluble protein (SP), free amino acid (FAA), and free fatty acid (FFA) in Chinese chive leaves were influenced by varying fungus gnat densities and CO2 concentrations (Fig. 2). Under the same population density, SS content increased with elevated CO2 (eCO2) concentration, though no significant differences were observed between ambient CO2 (aCO2) and eCO2 at 10 and 30 larvae/plant (Fig. 2A). In contrast, SP content decreased with increasing fungus gnat density under eCO2 (Fig. 2B), while FAA content showed an opposite trend, increasing with higher fungus gnat densities under eCO2 (Fig. 2C). FFA content was less affected by CO2 concentration than by fungus gnat infestation, displaying a general decreasing trend (Fig. 2D). At the same CO2 concentration, the highest SS content was observed at 30 larvae/plant, while the lowest occurred at 0 larvae/plant. Conversely, the highest levels of SP and FAA were found at 10 larvae/plant, with the lowest levels occurring at 30 larvae/plant.

Fig. 2
figure 2

Effects of eCO2 concentration and B. impatiens infestation on nutritional composition in Chinese chives. (A) Soluble sugar; (B) Soluble protein; (C) Free amino acid; (D) Free fatty acid. Asterisks of *, ** and ns respectively represent P < 0.05, P < 0.01, and P > 0.05 between two CO2 concentration. Lowercase letters of a, b and c denote significant differences (P < 0.05) among three infestation densities at 400 µL/L. Capital letters of A, B and C indicate significant differences (P < 0.05) among three infestation densities at 800 µL/L.

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Effects of eCO2 concentration and B. impatiens infestation on photosynthetic pigments in Chinese chives

Under elevated CO₂ (eCO2) conditions, the contents of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids in Chinese chive leaves were influenced by varying densities of B. impatiens infestation (Fig. 3). Compared to the 0 larvae/plant treatment, total chlorophyll content decreased significantly under both ambient CO2 (aCO2) and eCO2 conditions. At aCO2, the decrease was 20.89% and 41.60% for 10 and 30 larvae/plant, respectively, while under eCO2, the decrease was 20.06% and 38.58% for the same infestation levels (Fig. 3C). Similarly, carotenoid content declined under both CO2 conditions, with reductions of 12.36% and 14.29% at aCO2, and 5.09% and 28.92% at eCO2 for 10 and 30 larvae/plant, respectively, compared to the 0 larvae/plant treatment (Fig. 3D). Across all CO2 concentrations, the highest levels of chlorophyll a (Fig. 3A), chlorophyll b (Fig. 3B), total chlorophyll, and carotenoids were observed in the 0 larvae/plant treatment, while the lowest levels occurred at 30 larvae/plant.

Fig. 3
figure 3

Effects of eCO2 concentration and B. impatiens infestation on photosynthetic pigments in Chinese chives. (A) Chlorophyll a; (B) Chlorophyll b; (C) Total chlorophyll; (D) Carotenoid. Asterisks of *, ** and ns respectively represent P < 0.05, P < 0.01, and P > 0.05 between two CO2 concentration. Lowercase letters of a, b and c denote significant differences (P < 0.05) among three infestation densities at 400 µL/L. Capital letters of A, B and C indicate significant differences (P < 0.05) among three infestation densities at 800 µL/L.

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Correlation analysis

Correlation analysis revealed significant positive and negative relationships among plant growth parameters, nutritional composition and photosynthetic pigments under eCO2 stress and fungus gnat infestation (Fig. 4). Specifically, leaf width (LW) was positively correlated with plant height (PH) and soluble sugar (SS). Moreover, leaf thickness (LT) was positively correlated with SS, free amino acid (FFA) and Chlorophyll a (Ch-a). Plant height demonstrated a positive correlation with Ch-a content. Stem diameter (ST) was positively associated with soluble protein (SP) content, which in turn showed positive correlations with FAA and free fatty acid (FFA) contents. Additionally, total chlorophyll content exhibited positive correlations with both Ch-a and Chlorophyll b (Ch-b).

Fig. 4
figure 4

Correlation analysis among plant growth parameters, nutritional composition and photosynthetic pigments under eCO2 stress and fungus gnat infestation. LW: leaf width; LT: leaf thickness; PH: plant height; SD: stem diameter; SS: soluble sugar; SP: soluble protein; FAA: free amino acid; FFA: Free fatty acid; Ch-a: Chlorophyll a; Ch-b: Chlorophyll b; Ch: Chlorophyll; Ca: carotenoid. In the heatmap, colors closer to red indicate higher correlations, while colors closer to blue represent lower correlations.

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Discussion

Plant growth and development are significantly influenced by elevated CO2 (eCO2) concentrations, as evidenced by previous studies. eCO2 has been shown to enhance plant height and leaf area index in crops such as watermelon (Citrullus lanatus) and melon (Cucumis melo)20,21. Similarly, Li et al. (2013) reported an increase in the number of tillers in rice under eCO2 conditions22, while Zheng et al. (2019) observed a similar trend in soybean (Glycine max)23. In line with these findings, our study demonstrated that eCO2 led to increased leaf width and stem diameter in Chinese chive plants across varying B. impatiens infestation densities. These results suggest that eCO2 generally promotes vegetative growth, likely due to enhanced photosynthetic efficiency and carbon assimilation.

The nutritional composition of plants is also directly affected by CO2 concentrations. Hou et al., (2008) noted that eCO2 alters the carbon-to-nitrogen (C/N) ratio, increasing carbon content while decreasing nitrogen content24, a phenomenon supported by Sun et al. (2019)25. Our findings align with these observations, as soluble sugar content in Chinese chive increased under eCO2, while soluble protein content decreased, regardless of B. impatiens infestation levels. This is consistent with the results of Chen et al. (2006), who reported elevated glucose, total sugar and soluble sugar levels in spring wheat (Triticum aestivum) under eCO2, alongside reduced soluble protein content26. Similar trends were observed in cotton (Gossypium hirsutum), where eCO2 increased free amino acids and free fatty acids but decreased soluble protein27. Additionally, Chen et al. (2004) found that eCO2 increased glucose and total sugar content in wheat while reducing total nitrogen content28. In our study, free amino acid in Chinese chive leaves increased under eCO2 while free fatty acid content remained unaffected. These changes in nutrient composition may reflect a shift in plant metabolism under eCO2, favoring carbon-rich compounds over nitrogen-rich ones, which could have implications for plant-insect interactions and overall plant health.

Plants adapt to stress conditions, such as insect infestation, through various physiological mechanisms29. Stress can disrupt the photosynthetic apparatus, leading to reduced chlorophyll content30. Chlorophyll, a critical pigment for photosynthesis, plays a vital role in light absorption, energy transfer, and biomass accumulation, all of which influence plant growth and development31. In our study, B. impatiens infestation negatively impacted soluble protein, chlorophyll a, total chlorophyll and carotenoid in Chinese chive plants. The most significant reductions were observed at the highest infestation density (30 heads/plant) across both CO₂ concentrations. This aligns with findings from other plant species; for example, Wu et al. (2015) reported decreased soluble protein content in watermelon seeds following aphid infection32, while Najali et al. (2018) observed reduced chlorophyll content in plants damaged by the corn leaf aphid (Rhopalosiphum maidis)33. Similarly, Wójcicka et al. (2014) found that triticale seedlings infested with grain aphids (Sitobion avenae) exhibited lower chlorophyll and carotenoid levels but higher flavonoid content compared to uninfected plants34. These results suggest that insect infestation disrupts photosynthetic efficiency and alters secondary metabolite production, potentially as a defense mechanism.

The observed changes in plant growth and physiology under eCO2 and insect infestation can be attributed to several underlying mechanisms. Elevated CO₂ enhances photosynthesis, leading to increased carbohydrate production and biomass accumulation35. However, this often comes at the expense of nitrogen-based compounds, such as proteins, due to a shift in resource allocation. This shift may reduce the nutritional quality of plants for herbivores, potentially influencing insect feeding behavior and population dynamics.

Insect infestation, on the other hand, triggers stress responses in plants, including the production of secondary metabolites (e.g., flavonoids) and the degradation of chlorophyll, which may serve as defensive strategies36. The reduction in chlorophyll content under infestation likely reflects damage to the photosynthetic machinery, while the increase in free amino acids may indicate a breakdown of proteins into simpler nitrogenous compounds. These physiological changes could be part of a broader adaptive response to mitigate the impact of herbivory37.

In conclusion, our findings highlight the complex interplay between eCO2, insect infestation, and plant physiology. Elevated CO2 promotes growth and alters nutrient composition, while insect infestation induces stress responses that compromise photosynthetic efficiency and nutrient balance. Understanding these mechanisms is crucial for predicting how plants will respond to future climate scenarios and developing strategies to mitigate the impacts of pest infestations. Further research should focus on identifying specific secondary metabolites involved in plant defense and exploring their potential role in pest management under changing environmental conditions.

Conclusions

This study demonstrates that elevated CO2 (eCO2) and ambient CO2 (aCO2) conditions significantly influence the growth and physiological characteristics of Chinese chive plants, regardless of B. impatiens infestation. Specifically, leaf width, leaf thickness, soluble sugar content, and chlorophyll b levels showed a notable increase under both CO2 conditions. In contrast, soluble protein content, chlorophyll a, and total chlorophyll levels were significantly reduced. These findings highlight that B. impatiens infestation, combined with future eCO2 scenarios, can profoundly alter the physiological responses of Chinese chive plants. The study underscores the need for further research to identify secondary metabolites in the host plant that may play a role in defending against B. impatiens attacks. Additionally, investigating the presence and levels of compounds that could inhibit feeding by fungus gnat may provide critical insights for developing effective prevention and control strategies against B. impatiens. This research contributes to a deeper understanding of plant-insect interactions under changing atmospheric conditions and emphasizes the importance of developing adaptive strategies to mitigate the impacts of pest infestations in future climates.