Effects of nanoparticle applications on seedling survival and morphological characteristics in Scots pine afforestation

Abstract

This study was conducted in the afforestation area, using bare-root 2+0-year-old Scots pine seedlings from Kastamonu. The study aimed to determine the impact of nanoparticle (NP) applications on seedling morphological characteristics and seedling survival success. Three different concentrations (low, medium, high) and four different nanoparticle types [Fe₃O₄, CuO, ZnO, TiO₂] were applied to the plant root-dipping method in the study. The effects of NP treatments on seedling height (SH), root collar diameter (RCD), stem fresh weight (SFW), root new weight (RFW), seedling fresh weight (SEFW), root dry weight (RDW), stem dry weight (SDW), seedling dry weight (SEDW), sturdiness quotient (SI), root: shoot ratio (R/S), and seedling survival in the field were evaluated. The study results revealed that NP types significantly affected all seedling variables except RFW, SDW, RDW, and SEDW, and NP doses significantly affected all seedling variables except RFW. The binary interaction effects of NP types and doses had a significant effect on all seedling variables, and higher values were obtained compared to the control treatment. Medium and high NP doses were more effective in seedling growth than low doses; the percentage of seedling survival was 61.4% in the control treatment and 95% in the TiO₂-Medium NP treatment combination.

Full Article

Effects of Nanoparticle Applications on Seedling Survival and Morphological Characteristics in Scots Pine Afforestation

Sezgin Ayan,^a Esra Nurten Yer Çelik,^a Orhan Gülseven,^b,* Batın Mehmet Yer,^c and Muhammed Eskiömer ^d

This study was conducted in the afforestation area, using bare-root 2+0-year-old Scots pine seedlings from Kastamonu. The study aimed to determine the impact of nanoparticle (NP) applications on seedling morphological characteristics and seedling survival success. Three different concentrations (low, medium, high) and four different nanoparticle types [Fe₃O₄, CuO, ZnO, TiO₂] were applied to the plant root-dipping method in the study. The effects of NP treatments on seedling height (SH), root collar diameter (RCD), stem fresh weight (SFW), root new weight (RFW), seedling fresh weight (SEFW), root dry weight (RDW), stem dry weight (SDW), seedling dry weight (SEDW), sturdiness quotient (SI), root: shoot ratio (R/S), and seedling survival in the field were evaluated. The study results revealed that NP types significantly affected all seedling variables except RFW, SDW, RDW, and SEDW, and NP doses significantly affected all seedling variables except RFW. The binary interaction effects of NP types and doses had a significant effect on all seedling variables, and higher values were obtained compared to the control treatment. Medium and high NP doses were more effective in seedling growth than low doses; the percentage of seedling survival was 61.4% in the control treatment and 95% in the TiO₂-Medium NP treatment combination.

DOI: 10.15376/biores.18.4.8557-8572

Keywords: Afforestation; Seedling morphology; Scots pine; Nanoparticles

Contact information: a: Kastamonu University, Faculty of Forestry, Silviculture Department, Kastamonu, Türkiye; b: Kastamonu University, Institute of Science, Department of Forest Engineering, Kastamonu, Türkiye; c: Istanbul University-Cerrahpaşa, Faculty of Forestry, İstanbul, Türkiye; d: Kastamonu Forest Regional Directorate, Forest Management Directorate, Kastamonu, Türkiye;

* Corresponding author: gulsewenorhan@gmail.com

INTRODUCTION

Nanomaterials are structures with a size between 1 and 100 nm (Nel et al. 2006), and their strong physical and chemical properties enable them to be used in many fields (Rao et al. 2005; Gürmen and Ebin 2008; Ayan et al. 2021). These materials can be present in nature, and their concentrations are steadily increasing due to nanotechnological developments (Miller et al. 2004). People utilize metal-based nanoparticles (NPs) to make life easier across different sectors, and their use has rapidly grown in the last decade. Currently, the most popular NP structures are silver (Ag), as well as oxides of titanium (Ti), zinc (Zn), aluminum (Al), nickel (Ni), gold (Au), indium (In), molybdenum (Mo), copper (Cu), iron (Fe), bismuth (Bi), silica (Si), cobalt (Co), and tin (Sn). The most widely manufactured and commercially utilized metal-oxide NPs are titanium dioxide (TiO₂, zinc oxide (ZnO), iron oxide (Fe₃O₄), copper oxide (CuO), silicon dioxide (SiO₂), aluminum oxide (Al₂O₃), cerium dioxide (CeO₂), magnesium oxide (MgO), cuprous oxide (Cu₂O), nickel oxide (NiO), lanthanum oxide (La₂O₃), zirconium dioxide (ZrO₂), and indium oxide (In₂O₃) (Rajput et al. 2018). These materials have ecotoxicological effects, can accumulate in biological systems, and can be menacing when they undergo bioaccumulation and biodegradation in the food chain (Kuzma 2008). Moreover, nanoparticles (NPs) can cause different environmental impacts due to their physicochemical properties (Ma and Wang 2010). They have found wide application possibilities, particularly in medicine, pharmacy, construction, cosmetics, optics, and electronics sectors (Ruffini and Cremonini 2009; Kaweeteerawat et al. 2015; Tunca 2015).

In recent years, the effects of NP applications and how they are transported on plants have gained increasing importance in plant research (Du et al. 2011; Kundu et al. 2015). However, despite size exclusion limitations of 20 nm (Ma and Yan 2018; Ballikaya et al. 2022), it is commonly believed that particles > 100 nm can be taken up by plant roots. The results on root uptake vary depending on the kind of NPs, plant characteristics, and length of exposure (Ma and Wang 2010). According to Lv et al. (2019), stomata appear to be the main pathway for the foliar uptake of a wide variety of NPs. According to Avellan et al. (2021), the plant shape and physiological state (between 40 nm and 1 m) have an impact on the stomatal uptake of NPs. Nanoparticles promote plant growth by facilitating the uptake of nutrients from the soil because of their high surface volume and electronic structure, are used as fertilizers by increasing disease resistance, and are used in studies to prevent pests and diseases that infect plants (Servin et al. 2015). It is well known in this context that silver nanoparticles are extremely effective against a wide variety of fungi (Xue et al. 2016), bacteria (Rai et al. 2012), and viruses (Ardestani et al. 2015), including phytopathogenic ones. However, although silver nanoparticles (AgNP) in soil pose a significant environmental risk, there is little research on how such contamination may affect ectomycorrhizal fungi (EMFs) (Sweet and Singleton 2015).

Most studies suggest that NPs of 5 nm in size are taken up by both roots and aerial parts of plants and are transported through both the phloem and xylem (Dietz and Herth 2011; Wang et al. 2016; Ruttkay-Nedecky et al. 2017; Li et al. 2018). The NP applications, which are getting increasingly important, especially in agricultural studies, are being investigated for NP types and doses that will support breeding studies and ensure rapid germination and growth of seeds (Azura et al. 2017). The positive effects of pre-sowing treatment of wheat, maize, and rapeseed seeds with Cu and Zn NPs have been reported, but excess copper is toxic, and the root barrier plays an important role in the formation of tolerance to the surplus of this metal (Lebedev et al. 2016; Yausheva et al. 2017).

Moreover, the use of nanoparticles in forestry has become increasingly important in recent years. According to certain studies, mycorrhizal colonization of plant roots is negatively impacted (Dubchak et al. 2010), while other studies (Judy et al. 2015; Cao et al. 2017) contend that low nanoparticle concentrations have no effect on mycorrhization levels but that high concentrations have detrimental effects. Contrastingly, Feng et al. (2013) showed that AgNPs have a stimulatory effect on the development of arbuscular mycorrhiza, regardless of the used doses. According to Sweet and Singleton (2015), the variety of ectomycorrhizal fungi in the roots of Pinus muricata seedlings may drastically decrease as a result of soil contamination by AgNPs. Olchowik et al. (2017) found varying effects of metal nanoparticles on the type and concentration of ectomycorrhizae that form in pedunculate oak seedlings. Copper nanoparticles (Cu-NPs) were discovered to have a stimulatory effect at low concentrations but an inhibitory influence at high concentrations. Ag-NPs encouraged the formation regardless of concentration. In addition to the tree species, growth conditions, substrate type (soil or various nutrient media), temperature, and light intensity, their impact also appears to be influenced by the dosage, application method (foliar or soil), and concentration of the nanoparticles (Ruffini and Cremonini 2009). Zakharova et al. (2019) looked into how white poplar, crack willow, hairy birch, red oak, and pine micro clones adapted to ex vitro settings when exposed to zirconium trisulphide nanoplates and silver nanoparticles. The findings suggest that zirconium trisulphide nanoplates and silver nanoparticles have great potential for safeguarding woody species microclones when they are being transferred to non-sterile glasshouse environments. Additionally, because plants are known to be sensitive to nanoparticles, it is feasible that the presence of NP could have a direct impact on the growth of the trees (Yin et al. 2012). In addition, given the changes in climate, NP applications should be investigated to increase the seedling survival rate and to ensure better quality root and stem development in afforestation studies conducted in arid and semi-arid areas and that NP application can support forest tree breeding studies. Indeed, it is important for plants to benefit more from the soil in arid and semi-arid regions. For this purpose, using NPs in applications that increase drought resistance can be useful (Ashkavand et al. 2015). Reforestation studies for breeding trees resistant to ecological conditions in arid and semi-arid regions have become important to investigate the effectiveness of nanoparticles, especially in recent years when the effects of climate change have been intense. To this end, Ashkavand et al. (2015) suggested that nano silicon application on Crataegus sp. may be advantageous in reducing the harmful effects of drought stress. In Turkey, the development of seedlings was investigated by treating Anatolian black pine (Pinus nigra Arnold.) (Çelikbaş 2019), which is mainly used in the afforestation of semi-arid areas, Scots pine (Pinus sylvestris L.) (Çelikbaş 2019) and red pine (Pinus brutia Ten.) (Ayan et al. 2021) seeds with different NP types and doses before sowing, and the development of seedlings was investigated. It was reported that NP application was generally beneficial for black pine and Scots pine seeds germinated under laboratory conditions but did not contribute positively to red pine seeds sown under field conditions. Thus far, most of the research on the effect of nanoparticles on plants has been conducted in hydroponic culture (Bernhardt et al. 2010) or on peat-based substrates used in nursery production (Olchowik et al. 2017). Our hypothesis in this study is to examine the positive or negative effects of nanoparticle types and doses applied to the root area of saplings before planting on the development of saplings.

This study attempted to reveal the effects of applying different doses and types of nanoparticle solutions to the roots of seedlings before planting under field conditions on seedling survival rate and morphological development of seedlings in the field.

EXPERIMENTAL

In this research, NPs were added to the root zone of bare-rooted Scots pine seedlings. The effects of four different types of NP solutions at different dosage levels on seedling growth under field conditions were investigated using the root dipping method.

Materials

The bare-root 2+0 aged (2-year-old seedling produced by generative production method under nursery conditions) Scots pine seedlings originating from Araç-Dereyayla, Kastamonu were obtained from Kastamonu Taşköprü Forest Nursery. The experiment was established in section 191 of İhsangazi Forest Management Directorate, Mergüze Forest Management Chiefdom within the boundaries of Kapaklı Village (Coordinate: X 53° 89’50” – Y 45°53’13”.1 / 41°7’33”.91 N – 33°27’52”.97 E), which is 52.4 ha in size (Fig. 1).

Fig. 1. Study area

Fig. 2. General view of the research area

The site has an altitude of 1579 m with a southern facing and a slope of 31 to 60%. The average annual temperature of the region is 9.8 °C and the average rainfall is 482.3 mm. The soil structure of the site, which has sedimentary bedrock, is sandy-clay, and the seedlings were planted in the terraces established by edge planting at 1.5 × 1.5 m² intervals in April of 2019 (Fig. 2).

Methods

The doses specified in Table 1 were used in this study for the solutions of the most widely used NP types (Ashkavand et al. 2015; Rajput et al. 2018; Aleksandrowicz-Trzcińska et al. 2019; Khan et al. 2021).

Except for the control treatment, 30 seedlings were used for each NP solution and each different dose, and the experiment was established in a randomized block design with three replications. Using the root dipping method, the root zones of the seedlings were pretreated with NP solutions for 30 min before planting.

To determine the morphological characteristics of the seedlings in the dormant stage outside the vegetation period, the seedlings from the control treatment and each NP treatment group were uprooted in November 2019 without damaging the roots. Before uprooting, the root collar diameter of the seedlings was measured with a digital caliper with a precision of 0.1 mm, and the seedlings’ heights were measured with a steel tape measure with a precision of 1 mm. The roots of the seedlings were cleaned from the soil and individually tagged for determination of root/stem ratio and seedling fresh and dry weights. They were transferred to the laboratory for analysis.

Table 1. Types and Doses of Used Nanoparticles

Measurements and calculations made in the laboratory

In the seedlings brought to the laboratory in November to December 2019, seedling root collar diameter (RCD-mm) was determined with 0.1 mm precision from the root collar, seedling height (SH-cm) was determined with 0.1 cm precision from the root collar diameter to the terminal shoot tip, and the sturdiness index (SI) was calculated as the ratio of SH to RCD (Aphalo and Rikala 2003) as given below in Eq. 1:

(1)

The parts of the seedlings above and below the RCD were measured with a precision of 0.001 g. The weight values were stem fresh weight (SFW-g) and root fresh weight (RFW-g), the sum of these two values was seedling fresh weight (SEFW-g), while stem dry weight (SDW-g), root dry weight (RDW-g), and seedling dry weight (SEDW-g) were determined by drying these parts at 101 +/- 4 °C for 24 h. The root:shoot ratio (R/S) index was calculated as the ratio of stem dry weight to root dry weight (Ayan 2002), given as Eq. 2:

(2)

Statistical Evaluations

Multivariate analysis of variance was applied to determine whether RCD, SH, SFW, RFW, SEFW, SDW, RDW, SEDW, SI, and R/S variables of seedlings showed significant differences in terms of various NP treatments and different doses, and homogeneous groups were determined by Duncan test. All data were analyzed with IBM SPSS 23 software (IBM Corp., Armonk, NY, USA) at a p = 0.05 significance level.

RESULTS AND DISCUSSION

The significant effects of NP types applied to Scots pine seedlings on all other variables except RFW, SDW, RDW, and SEDW variables are shown in Table 2. According to the general average values given in Table 2, the lowest values were in the control group in all studied seedling variables. While the TiO₂ NP solution was the most efficient in SH development, the lowest SH development in NP-treated seedlings was the ZnO solution. In terms of root collar diameter development, CuO, and TiO₂ were the most effective, while ZnO had the lowest effect. CuO NP application had the most positive effect on SFW, SDW, and SEFW variables. In terms of SI and R/S, it was determined that the seedlings allocated as control were more advantageous (Table 2).

The NP doses had a significant effect on all seedling variables except RFW (Table 3). Compared to the control treatment, all NP dose treatments had a positive effect on seedling fresh and dry weight values except SI and R/S. Among the doses, “Medium” and “High” amounts were more efficient than low doses (Table 3).

The highest values on the SH variable were detected in TiO₂-Medium (21.42 ± 0.93 cm) and CuO-High (21.38 ± 1.05 cm) dose applications. The effects of the same nanoparticles were also found to be high on the RCD [TiO₂-Medium (6.83 ± 0.27 mm), CuO-High (6.83 ± 0.32 mm)] variable. When Table 4 is examined, the ZnO-Medium treatment combination on the following variables [SFW (17.42 ± 4.34 g), RFW (6.06 ± 1.49 g), SEFW (23.47 ± 5.82 g), SDW (7.75 ± 1.98 g), RDW (7.75 ± 1.97 g) and SEDW (10.69 ± 2.71 g)] supported plant growth in the most positive way. The control treatment (20.01 ± 0.09) had the most favorable value in the SI variable, and the TiO₂-Low (2.15 ± 0.11) treatment combination had the best value in the R/S variable (Table 4).

It was determined that NP treatments generally had a positive effect on the survival rate of seedlings compared to the control treatment (61.39%), and the highest survival rate was obtained with TiO₂ NP (95%) applied at a medium dose (Table 5).

Table 2. Analysis of Variance and Duncan Test Results for the Effect of NP Type

Table 3. Analysis of Variance and Duncan Test Results for the Effect of NP Dose

Table 4. Analysis of Variance and Duncan Test Results for the Binary Interactions of NP Type and Dose Factors

Table 5. Statistics on the Interactive Effect of Nanoparticle Type and Dose on Seedling Survival