Nanotechnology enables precise delivery, controlled release, and real-time tracking of bioactive compounds in plant systems, offering powerful opportunities for sustainable crop improvement and advanced plant biotechnology. Cellular internalization of nanomaterials (NMs) in plants may occur via transient pore formation, carrier-mediated transport, or interactions with membrane proteins such as aquaporins and ion channels. Growing evidence indicates that endocytosis-like mechanisms are key contributors to NM uptake in plant cells. In this review, we collect and critically discuss the role of endocytic pathways in NM uptake by plant cells, in addition to identifying knowledge gaps and future research directions. Many studies show that clathrin-mediated endocytosis (CME) plays a major role in NM internalization, however, clathrin-independent pathways may also contribute depending on the plant–NM system. Uptake efficiency is influenced by NM properties, including size, charge, and surface functionalization, as well as plant-specific factors such as cell wall architecture and endocytic capacity. Redox signaling mediated by nitric oxide and reactive oxygen species regulates endocytic machinery, thereby shaping NM uptake efficiency. Conversely, NMs can modify cellular redox homeostasis, creating bidirectional feedback that determines their intracellular fate. Endocytic NM uptake intersects with nutrient transport and stress-signaling networks, enabling NMs to modulate antioxidant defenses, hormone signaling, and root developmental plasticity, while potentially competing with micronutrient transporters. This perspective suggests that endocytosis functions as a regulated interface between NMs and plant cells, where nanoparticle properties and cellular redox processes together shape their uptake, trafficking, and biological effects.

Hemiparasitic plants are known to be able to diversify degraded grasslands by lowering the competitive power of dominant grasses. Recent research indicates that hemiparasites may also be used against invasive alien plants. Here, we tested the effects of Odontites luteus, a native European hemiparasite, on Sporobolus cryptandrus, a recently established and rapidly spreading C4 grass of North-American origin. We found that Odontites considers Sporobolus a suitable host and reduces its biomass production (and potentially its competitive ability) by approx. 50%, equaling the effect on its major native host, Festuca vaginata. However, Festuca showed severe metabolic impairment (reduced photosynthetic capacity and increased physiological stress) under hemiparasite pressure. So, the application of hemiparasites is a promising biocontrol tool against Sporobolus (and potentially other invasive C4 grasses), but it is not a silver bullet. We cannot expect a full eradication of the invasive species and a recovery of the native community but thinning monodominant Sporobolus stands to allow certain populations of native species, particularly those resistant to Odontites, to come back is a more realistic goal. This could lead to a partial recovery of the former species composition and an improvement of ecosystem functions, such as providing food for pollinators.

The interaction between ethylene (ET) and light/dark cycles is complex and regulates plant growth, development and stress responses. While ET synthesis is influenced by circadian rhythms, light -especially red light- it can also affect ET-regulated processes, depending on the developmental stage and organ type. This study investigated the effects of nocturnal red light exposure on mature tomato plant leaves, using wild-type (WT) and ET-insensitive Never ripe (Nr) mutant. Red light application for one week at night did not significantly affect leaf biomass and area, but it altered structural traits such as leaf thickness and internode length, particularly in WT plants. Transcriptomic analyses indicated significant differences in gene expression between WT and Nr plants, particularly in genes related to photosynthesis, growth regulation and the circadian clock. Red light also modulated the expression of hormone-related genes and increased the levels of defence-related phytohormones (ET, salicylic acid, abscisic acid) as well as the activity of antioxidant enzymes (e.g. guaiacol peroxidase, glutathione S-transferase) in a manner dependent on ET in WT plants. Interestingly, although ET is involved in many of these processes, red light was also found to increase biotic stress resistance in Nr mutants, as evidenced by reduced Botrytis cinerea infection. This suggests that red light may activate defence pathways independently of ET. Our findings highlight the potential of red light as an environmentally friendly tool for improving crop disease resistance and emphasise the pivotal role of ET signalling, which can be regulated by specific light spectra, in plant’s defence reactions.

Bacterial flagellin (flg22) induces rapid and permanent stomatal closure. However, its local and systemic as well as tissue- and cell-specific effects are less understood. Our results show that flg22 induced local and systemic stomatal closure in intact tomato plants, which was regulated by reactive oxygen- and nitrogen species, and also affected the photosynthetic activity of guard cells but not of mesophyll cells. Interestingly, rapid and extensive local expression of Ethylene response factor 1 was observed after exposure to flg22, whereas the relative transcript levels of Defensin increased only after six hours, especially in systemic leaves. Following local and systemic ethylene emission already after one and six hours, jasmonic acid levels increased in the local leaves after six hours of flg22 treatment. Using immunohistochemical methods, significant defensin accumulation was found in the epidermis and stomata of flg22-treated leaves and above them. Immunogold labelling revealed significant levels of defensins in the cell wall of the mesophyll parenchyma and guard cells. Furthermore, single cell qRT-PCR confirmed that guard cells are able to synthesise defensins. It can be concluded that guard cells are not only involved in the first line of plant defense by regulating stomatal pore size, but can also defend themselves and the plant by producing and accumulating antimicrobial defensins where phytopathogens can penetrate.

This study explores the long-term effects of nocturnal red light treatment on tomato plants, focusing on photosynthesis, metabolism, and defence mechanisms. The impact of red light treatment applied at night to both young and old leaves at various times of day was investigated. The results showed that, after three weeks, nocturnal red light treatment improved photosynthetic efficiency in both upper and lower leaves, particularly in the morning and at noon. This enhancement in photosynthesis was associated with higher CO2 assimilation. Interestingly, older leaves, which usually demonstrate reduced photosynthetic activity, also benefited from red light exposure. Additionally, red light treatment resulted in significant changes to the accumulation of starch and sugars. Higher levels of starch were found in the leaves in the afternoon, alongside increased levels of sucrose in both leaves. Metabolomics analysis revealed that the level of several defence-related metabolites, including 4-guanidinobutyric acid, fucose and quinic acid, increased following exposure to red light. This suggests that red light applied at night could have a priming effect, enhancing defence against pathogens. Conversely, the levels of some metabolites, such as myo-inositol and N-acetylaspartate, decreased, indicating adjustments in plant stress responses. Red light influenced plant height and leaf area and enhanced callose content in older leaves. Moreover, red light can hard plants against fungal pathogens, such as Botrytis cinerea. These results suggest that exposure to red light at night is an effective, environmentally friendly approach to enhance the resilience of tomato plants and optimise greenhouse farming practices, particularly through supplemental or interlighting systems.

Background and aimsSmoking is recognised as an independent risk factor in the development of chronic pancreatitis (CP). Cystic fibrosis transmembrane conductance regulator (CFTR) function and ductal fluid and bicarbonate secretion are also known to be impaired in CP, so it is crucial to understand the relationships between smoking, pancreatic ductal function and the development of CP.MethodsWe measured sweat chloride (Cl-) concentrations in patients with and without CP, both smokers and non-smokers, to assess CFTR activity. Serum heavy metal levels and tissue cadmium concentrations were determined by mass spectrometry in smoking and non-smoking patients. Guinea pigs were exposed to cigarette smoke, and cigarette smoke extract (CSE) was prepared to characterise its effects on pancreatic HCO3- and fluid secretion and CFTR function. We administered cerulein to both the smoking and non-smoking groups of mice to induce pancreatitis.ResultsSweat samples from smokers, both with and without CP, exhibited elevated Cl- concentrations compared to those from non-smokers, indicating a decrease in CFTR activity due to smoking. Pancreatic tissues from smokers, regardless of CP status, displayed lower CFTR expression than those from non-smokers. Serum levels of cadmium and mercury, as well as pancreatic tissue cadmium, were increased in smokers. Smoking, CSE, cadmium, mercury and nicotine all hindered fluid and HCO3- secretion and CFTR activity in pancreatic ductal cells. These effects were mediated by sustained increases in intracellular calcium ([Ca2+]i), depletion of intracellular ATP (ATPi) and mitochondrial membrane depolarisation.ConclusionSmoking impairs pancreatic ductal function and contributes to the development of CP. Heavy metals, notably cadmium, play a significant role in the harmful effects of smoking.Key points Smoking and cigarette smoke extract diminish pancreatic ductal fluid and HCO3- secretion as well as the expression and function of CFTR Cd and Hg concentrations are significantly higher in the serum samples of smokers Cd accumulates in the pancreatic tissue of smokers Smoking and cigarette smoke extract diminish pancreatic ductal fluid and HCO3- secretion as well as the expression and function of CFTR Cd and Hg concentrations are significantly higher in the serum samples of smokers Cd accumulates in the pancreatic tissue of smokers image

Fusarium pathogens are causal agents of several crop diseases and produce harmful mycotoxins resulting in crop and yield reduction worldwide. Among crop diseases, Fusarium wilt, Fusarium head blight, and Fusarium root blight are mostly reported diseases in numerous vegetables, crops, and fruits and have posed pressure on current food production and safety. In addition, the production of mycotoxins further aggravates plant health and causes serious health risks in humans and animals through food chain contamination. Different management practices have been enlisted in this chapter to reduce or eradicate Fusarium wilt in different crops. Interestingly, various mechanisms developed by plants have also been highlighted to fight against Fusarium pathogens and limit the growth of mycotoxins. One of defence mechanisms is plant antioxidant mechanisms to reduce oxidative stress by increasing enzymatic and non-enzymatic antioxidants to maintain cellular homeostasis under Fusarium infection. The other defence response is through hormonal signalling to combat fungal pathogens. Different phytohormones such as salicylic acid, ethylene, jasmonate, abscisic acid, cytokinin, auxin, and other plant secondary metabolites play a crucial part in the reduction of Fusarium growth and inhibit mycotoxin production through defence-related genes. Further, the use of different pre-harvest and post-harvest strategies has been elucidated to enhance plant resistance and growth by decreasing fungal pathogenicity and virulence.

Plant infections caused by fungi lead to significant crop losses worldwide every year. This study aims to better understand the plant defence mechanisms regulated by red light, in particular, the effects of red light at night when most phytopathogens are highly infectious. Our results showed that superoxide production significantly increased immediately after red light exposure and, together with hydrogen peroxide levels, was highest at dawn after 30 min of nocturnal red-light treatment. In parallel, red-light-induced expression and increased the activities of several antioxidant enzymes. The nocturnal red light did not affect salicylic acid but increased jasmonic acid levels immediately after illumination, whereas abscisic acid levels increased 3 h after nocturnal red-light exposure at dawn. Based on the RNAseq data, red light immediately increased the transcription of several chloroplastic chlorophyll a-b binding protein and circadian rhythm-related genes, such as Constans 1, CONSTANS interacting protein 1 and zinc finger protein CONSTANS-LIKE 10. In addition, the levels of several transcription factors were also increased after red light exposure, such as the DOF zinc finger protein and a MYB transcription factor involved in the regulation of circadian rhythms and defence responses in tomato. In addition to identifying these key transcription factors in tomato, the application of red light at night for one week not only reactivated key antioxidant enzymes at the gene and enzyme activity level at dawn but also contributed to a more efficient and successful defence against Botrytis cinerea infection.

Plant hormones such as ethylene (ET) and salicylic acid (SA) have an elementary role in the regulation of ER stress and unfolded protein response (UPR) in plants via modulating defence responses or inducing oxidative stress. Chloroplasts can be sources and targets of reactive oxygen species (ROS) that affect photosynthetic efficiency, which has not been investigated under tunicamycin (Tm)-induced ER stress. In this study, the direct and indirect effects of Tm on chloroplastic ROS production were first investigated in leaves of wild-type tomato (Solanum lycopersicum L.) plants. Secondly changes in activities of photosystem II and I were analysed under Tm exposure and after application of the chemical chaperone 4-phenylbutyrate (PBA) in different genotypes, focusing on the regulatory role of SA and ET Tm treatments significantly but indirectly induced ROS production in tomato leaves and in parallel it decreased the effective quantum yield of PSII [Y(II)] and PSI [Y(I)], as well as the photochemical quenching coefficient (qP) and the quantum yield of non-photochemical energy dissipation in PSI due to acceptor-side limitation [Y(NA)]. At the same time, Tm increased non-photochemical quenching (NPQ) and cyclic electron flow (CEF) in tomato leaves after 24 h. However, the photosynthetic activity of the SA hydroxylase-overexpressing NahG tomato plants was more severely affected by Tm as compared to wild-type and ET-insensitive Never ripe (Nr) plants. These results suggest the protective role of SA in the regulation of photosynthetic activity contributing to UPR and the survival of plants under ER stress. Interestingly, the activation of photoprotective mechanisms by NPQ was independent of SA but dependent on active ET signalling under ER stress, whereas CEF was reduced by ET due to its higher ratio in Nr plants.

Plant defence responses induced by the bacterial elicitor flg22 are highly dependent on phytohormones, including gaseous ethylene (ET). While the regulatory role of ET in local defence responses to flg22 exposure has been demonstrated, its contribution to the induction of systemic responses is not clearly understood. For this consideration, we examined the effects of different ET modulators on the flg22-induced local and systemic defence progression. In our experiments, ET biosynthesis inhibitor aminoethoxyvinyl glycine (AVG) or ET receptor blocker silver thiosulphate (STS) were applied 1 h before flg22 treatments and 1 h later the rapid local and systemic responses were detected in the leaves of intact tomato plants (Solanum lycopersicum L.). Based on our results, AVG not only diminished the flg22-induced ET accumulation locally, but also in the younger leaves confirming the role of ET in the whole-plant expanding defence progression. This increase in ET emission was accompanied by increased local expression of SlACO1, which was reduced by AVG and STS. Local ET biosynthesis upon flg22 treatment was shown to positively regulate local and systemic superoxide (O2.-) and hydrogen peroxide (H2O2) production, which in turn could contribute to ET accumulation in younger leaves. Confirming the role of ET in flg22-induced rapid defence responses, application of AVG reduced local and systemic ET, O2.and H2O2 production, whereas STS reduced it primarily in the younger leaves. Interestingly, in addition to flg22, AVG and STS induced stomatal closure alone at whole-plant level, however in the case of combined treatments together with flg22 both ET modulators reduced the rate of stomatal closure in the older- and younger leaves as well. These results demonstrate that both local and systemic ET production in sufficient amounts and active ET signalling are essential for the development of flg22-induced rapid local and systemic defence responses.

The mycotoxin fusaric acid (FA) induces rapid oxidative burst leading to cell death in plants. At the same time, plant defence reactions are mediated by several phytohormones for instance ethylene (ET). However, previously conducted studies leave research gaps on how ET plays a regulatory role under mycotoxin exposure. Therefore, this study aims to the time-dependent effects of two FA concentrations (0.1 mM and 1 mM) were explored on the regulation of reactive oxygen species (ROS) in leaves of wild-type (WT) and ET receptor mutant Never ripe (Nr) tomatoes. FA induced superoxide and H2O2 accumulation in both genotypes in a mycotoxin dose-and exposure time-dependent pattern. 1 mM FA activated NADPH oxidase (+34% compared to the control) and RBOH1 transcript levels in WT leaves. However, superoxide production was significantly higher in Nr with 62% which could contribute to higher lipid peroxidation in this genotype. In parallel, the antioxidative defence mechanisms were also activated. Both peroxidase and superoxide dismutase activities were lower in Nr but ascorbate peroxidase showed one-fold higher activity under 1 mM FA stress than in WT leaves. Interestingly, catalase (CAT) activity decreased upon FA in a time-and concentration-dependent manner and the encoding CAT genes were also downregulated, especially in Nr leaves at 20%. Ascorbate level was decreased and glutathione remained lower in Nr than WT plants under FA exposure. Conclusively, Nr genotype showed more sensitivity to FA-induced ROS suggesting that ET serves defence reactions of plants by activating several enzymatic and non-enzymatic antioxidants to detoxify excess ROS accumulation.

Fusaric acid (FA) is one of the most harmful phytotoxins produced in various plant–pathogen interactions. Fusarium species produce FA as a secondary metabolite, which can infect many agronomic crops at all stages of development from seed to fruit, and FA production can further compromise plant survival because of its phytotoxic effects. FA exposure in plant species adversely affects plant growth, development and crop yield. FA exposure in plants leads to the generation of reactive oxygen species (ROS), which cause cellular damage and ultimately cell death. Therefore, FA-induced ROS accumulation in plants has been a topic of interest for many researchers to understand the plant–pathogen interactions and plant defence responses. In this study, we reviewed the FA-mediated oxidative stress and ROS-induced defence responses of antioxidants, as well as hormonal signalling in plants. The effects of FA phytotoxicity on lipid peroxidation, physiological changes and ultrastructural changes at cellular and subcellular levels were reported. Additionally, DNA damage, cell death and adverse effects on photosynthesis have been explained. Some possible approaches to overcome the harmful effects of FA in plants were also discussed. It is concluded that FA-induced ROS affect the enzymatic and non-enzymatic antioxidant system regulated by phytohormones. The effects of FA are also associated with other photosynthetic, ultrastructural and genotoxic modifications in plants. Graphical abstract

Fumonisin B1 (FB1) is a harmful mycotoxin produced by Fusarium species, which results in oxidative stress leading to cell death in plants. FB1 perturbs the metabolism of sphingolipids and causes growth and yield reduction. This study was conducted to assess the role of ethylene in the production and metabolism of reactive oxygen species in the leaves of wild type (WT) and ethylene receptor mutant Never ripe ( Nr ) tomato and to elucidate the FB1-induced phytotoxic effects on the photosynthetic activity and antioxidant mechanisms triggered by FB1 stress. FB1 exposure resulted in significant ethylene emission in a concentration-dependent manner in both genotypes. Moreover, FB1 significantly affected the photosynthetic parameters of PSII and PSI and activated photoprotective mechanisms, such as non-photochemical quenching in both genotypes, especially under 10 µM FB1 concentration. Further, the net photosynthetic rate and stomatal conductance were significantly reduced in both genotypes in a FB1 dose-dependent manner. Interestingly, lipid peroxidation and loss of cell viability were also more pronounced in WT as compared to Nr leaves indicating the role of ethylene in cell death induction in the leaves. Thus, FB1-induced oxidative stress affected the working efficiency of PSI and PSII in both tomato genotypes. However, ethylene-dependent antioxidant enzymatic defense mechanisms were activated by FB1 and showed significantly elevated levels of superoxide dismutase (18.6%), ascorbate peroxidase (129.1%), and glutathione S-transferase activities (66.62%) in Nr mutants as compared to WT tomato plants confirming the role of ethylene in the regulation of cell death and defense mechanisms under the mycotoxin exposure.

Flagellin (flg22) induces rapid and long-lasting defence responses. It may also affect the photosynthetic activity depending on several internal and external factors, such as the phytohormone ethylene or the day/night time. Based on the results, flg22 treatment, neither in the light phase nor in the evening, caused any significant change in chlorophyll fluorescence induction parameters in the leaves of wild-type and ethylene-receptor mutant Never ripe tomato plants measured the next morning. However, flg22 in the light phase decreased the effective quantum yield and the photochemical quenching both locally and systemically in guard cells. In parallel, the production of reactive oxygen species and nitric oxide increased, which contributed to the stomatal closure and a decrease in CO2 assimilation the next day. A decrease in sugar content and elevated hexokinase activity measured after flg22 exposure can also contribute to local defence responses in intact tomato plants.

Plant pathogenic fungi are responsible for enormous crop losses worldwide. Overcoming this problem is challenging as these fungi can be highly resistant to approved chemical fungicides. There is thus a need to develop and introduce fundamentally new plant and crop protection strategies for sustainable agricultural production. Highly stable extracellular antifungal proteins (AFPs) and their rationally designed peptide derivatives (PDs) constitute feasible options to meet this challenge. In the present study, their potential for topical application to protect plants and crops as combinatorial biofungicides is supported by the investigation of two Neosartorya (Aspergillus) fischeri AFPs (NFAP and NFAP2) and their γ-core PDs. Previously, the biofungicidal potential of NFAP, its rationally designed γ-core PD (γNFAP-opt), and NFAP2 was reported. Susceptibility tests in the present study extended the in vitro antifungal spectrum of NFAP2 and its γ-core PD (γNFAP2-opt) to Botrytis, Cladosporium, and Fusarium spp. Besides, in vitro additive or indifferent interactions, and synergism were observed when NFAP or NFAP2 was applied in combination with γNFAP-opt. Except for γNFAP2-opt, the investigated proteins and peptides did not show any toxicity to tomato plant leaves. The application of NFAP in combination with γNFAP-opt effectively inhibited conidial germination, biofilm formation, and hyphal extension of the necrotrophic mold Botrytis cinerea on tomato plant leaves. However, the same combination only partially impeded the B. cinerea-mediated decay of tomato fruits, but mitigated the symptoms. Our results highlight the feasibility of using the combination of AFP and PD as biofungicide for the fungal infection control in plants and crops.

The first line of plant defence responses against pathogens can be induced by the bacterial flg22 and can be dependent on various external and internal factors. Here, we firstly studied the effects of daytime and ethylene (ET) using Never ripe (Nr) mutants in the local and systemic defence responses of intact tomato plants after flg22 treatments. Flg22 was applied in the afternoon and at night and rapid reactions were detected. The production of hydrogen peroxide and nitric oxide was induced by flg22 locally, while superoxide was induced systemically, in wild type plants in the light period, but all remained lower at night and in Nr leaves. Flg22 elevated, locally, the ET, jasmonic acid (JA) and salicylic acid (SA) levels in the light period; these levels did not change significantly at night. Expression of Pathogenesis-related 1 (PR1), Ethylene response factor 1 (ERF1) and Defensin (DEF) showed also daytime- and ET-dependent changes. Enhanced ERF1 and DEF expression and stomatal closure were also observable in systemic leaves of wild type plants in the light. These data demonstrate that early biotic signalling in flg22-treated leaves and distal ones is an ET-dependent process and it is also determined by the time of day and inhibited in the early night phase.

EDITORIAL article Front. Plant Sci., 28 May 2021 | https://doi.org/10.3389/fpls.2021.688053

Plant defence responses can be triggered by the application of elicitors for example chitosan (β-1,4-linked glucosamine; CHT). It is well-known that CHT induces rapid, local production of reactive oxygen species (ROS) and nitric oxide (NO) resulting in fast stomatal closure. Systemic defence responses are based primarily on phytohormones such as ethylene (ET) and salicylic acid (SA), moreover on the expression of hormone-mediated defence genes and proteins. At the same time, these responses can be dependent also on external factors, such as light but its role was less-investigated. Based on our result in intact tomato plants (Solanum lycopersicum L.), CHT treatment not only induced significant ET emission and stomatal closure locally but also promoted significant production of superoxide which was also detectable in the distal, systemic leaves. However, these changes in ET and superoxide accumulation were detected only in wild type (WT) plants kept in light and were inhibited under darkness as well as in ET receptor Never ripe (Nr) mutants suggesting pivotal importance of ET and light in inducing resistance both locally and systemically upon CHT. Interestingly, CHT-induced NO production was mostly independent of ET or light. At the same time, expression of Pathogenesis-related 3 (PR3) was increased locally in both genotypes in the light and in WT leaves under darkness. This was also observed in distal leaves of WT plants. The CHT-induced endoplasmic reticulum (ER) stress, as well as unfolded protein response (UPR) were examined for the first time, via analysis of the lumenal binding protein (BiP). Whereas local expression of BiP was not dependent on the availability of light or ET, systemically it was mediated by ET.

Selenium (Se) enrichment of Stevia rebaudiana Bertoni can serve a dual purpose, on the one hand to increase plant biomass and stress tolerance and on the other hand to produce Se fortified plant-based food. Foliar Se spraying (0, 6, 8, 10 mg/L selenate, 14 days) of Stevia plantlets resulted in slightly decreased stevioside and rebaudioside A concentrations, and it also caused significant increment in stem elongation, leaf number, and Se content, suggesting that foliar Se supplementation can be used as a biofortifying approach. Furthermore, Se slightly limited photosynthetic CO2 assimilation (AN, gsw, Ci/Ca), but exerted no significant effect on chlorophyll, carotenoid contents and on parameters associated with photosystem II (PSII) activity (FV/FM, F0, Y(NO)), indicating that Se causes no photodamage in PSII. Further results indicate that Se is able to activate PSI-cyclic electron flow independent protection mechanisms of the photosynthetic apparatus of Stevia plants. The applied Se activated superoxide dismutase (SOD) isoenzymes (MnSOD1, FeSOD1, FeSOD2, Cu/ZnSOD1, Cu/ZnSOD2) and down-regulated NADPH oxidase suggesting the Se-induced limitation of superoxide anion levels and consequent oxidative signalling in Stevia leaves. Additionally, the decrease in S-nitrosoglutathione reductase protein abundance and the intensification of protein tyrosine nitration indicate Se-triggered nitrosative signalling. Collectively, these results suggest that Se supplementation alters Stevia shoot morphology without significantly affecting biomass yield and photosynthesis, but increasing Se content and performing antioxidant effects, which indicates that foliar application of Se may be a promising method in Stevia cultivation.