Yield, volume, quality, and reduction of biotic stress in ﬂ uenced by titanium application in oilseed rape, winter wheat, and maize cultivations

: The study presents the results of research on the in ﬂ uence of a mineral growth stimulant containing titanium ( Ti ) in the form available to plants, applied to reduce the e ﬀ ects of biotic stresses caused by agro -phages, namely fungal pathogens and selected insect pests. The study was conducted in 2014 and 2015 on winter oilseed rape, winter wheat, and maize. The pur pose of the study was to determine the in ﬂ uence of the Ti containing stimulant on the degree of damage caused by major pests occurring in the crops ( cabbage seed weevil, cereal leaf beetle, and European corn borer ) , the degree of infestation with fungal diseases ( gray mold, Alternaria disease, eyespot, foot rot, sooty mold, glume blotch, Fusarium head blight, Fusarium stalk rot, maize smut, and brown spot ) , and yield parameters. The study showed that the stimulant containing Ti successfully reduced the occurrence of pest damage to winter rapeseed and winter wheat plants and the occurrence of diseases in winter rapeseed, winter wheat, and maize crops. Thus, the appli cation of the Ti stimulant resulted in an increased yield of the crops being tested. The main factor explaining this phenomenon is unknown, and it is probably the result of several factors. The study contains the discussion on this phenomenon.


Introduction
In addition to fungicides, herbicides, and insecticides, today's agriculture uses a number of agents classified as plant growth stimulants or biostimulants. Their role involves the regulation and acceleration of life processes, improvement of plant resistance to stress factors, and stimulation of root and leaf development. Due to their mode of action, these substances are safe for the natural environment and thus partially substitute chemical plantprotection agents. The most common groups of substances stimulating plant growth and life processes include phytohormones (e.g., auxins, cytokinins, and gibberellins), bioregulators (e.g., phenols and salicylic acid), biostimulants (also called growth stimulants; phytostimulants), antagonists (fungi and bacteria), and substances, which have an indirect effect on plants through the soil environment and its improvement (soil improvers). Titanium (Ti) belongs to the biostimulant group [1].
Ti is a chemical element of the metal group. It is relatively common in soil, in the form of titanium oxide (TiO 2 ; 0.5-1.5%; on an average of 0.61%). The largest amounts of Ti can be found in volcanic ash [2]. The availability of Ti to plants is strictly connected with the type of soil. It increases along with a decrease in the soil pH and increases in the proportion of organic matter. In Poland, the content of Ti in the soil is less than 1%. The smallest amounts of this element can be found in peaty soils (about 0.05%), whereas the largest amounts are in loamy soils and rendsina soils (about 0.8%) [3]. In spite of the common occurrence of Ti in soil, its ions are not easily accessible to plants. The influence of Ti on plants has been studied for many years. As early as the 1930s, research on Ti content in crops revealed a dependence between this element and silicon (Si). The higher the Si content in plants, the easier it is for them to take up Ti. Similar to Si, Ti exhibits many properties stimulating plants [3][4][5]. So far, a few mechanisms of stimulating plants have been investigated. However, other aspects have not been investigated, and there are still studies being done to determine the mechanisms of action of Ti ions in plants.
Ti ions intensify the activity of iron ions in plants. This dependence is closely correlated with chlorophyll synthesis, photosynthesis intensity, and the activity of enzymes responsible for the elimination of free radicals, such as peroxidase, catalase, nitrogenase, and nitrate reductase [5]. These effects are particularly important in stressful situations as the plant is much less influenced by the consequences of stress caused by low temperatures, a deficit of light and water, or low activity of the root system. Another important advantage of Ti ions is the fact that they stimulate the uptake of nutrients provided to plants from the soil solution. In consequence, the osmotic pressure in the aerial part of the plant increases. As the plant tries to equalise the pressure, it stimulates the root system to take up more water and nutrients [4]. One of the major advantages of Ti ions is the fact that they increase pollen vitality and positively influence the processes of pollination and fertilization [6]. This advantage is becoming increasingly important in view of the fact that the number of pollinating insects is decreasing dramatically due to chemicals used in agriculture. It has been observed that the application of Ti to plants limits the occurrence of some fungal and bacterial diseases and reduces the level of damage caused by pests [7]. Stimulants containing Ti are known to have direct, positive effects on plants as they increase their size (biomass) and boost plants' natural resistance to biotic stresses such as insect pests and fungal diseases. They improve the quantitative and qualitative parameters of the yield and thus increase the yield [7][8][9]. Their mechanism of action consists in accelerating the uptake of nutrients, increasing the activity of iron ions, and increasing the pollen tube receptivity and pollen vitality [10][11][12]. The aim of this study was to test the possibility of reducing the amount of insect damage and the level of fungal infestation in the aforementioned crops with the use of a mineral growth stimulant containing Ti in the form available to plants. The article presents the results of 2 year experiments on the influence of the liquid biostimulant "Tytanit," which contains 8.5 g of Ti ions per liter, on winter oilseed rape, winter wheat, and maize. The soil had the following characteristics: soil type: argillaceous sand, soil quality class: IIIa, organic matter: 1.14%, and soil pH: 4.9. Oilseed rape was sown on  The crops were fully protected with pesticides.

Materials and methods
The following fungicides containing active ingredients from the group of triazoles, strobilurins, and ketoamines were applied in the experiments: The following insecticides containing active ingredients from the group of pyrethroids and neonicotinoids were applied in the experiments: There were no insecticidal treatments in maize plantations.
The plants were treated with a field backpack sprayer, with a 50 cm boom (sprayer volume 6.0 L, nozzle ID: 4 TeeJet XR11002VSnozzles), at a wind speed of less than 2 km/h. The first treatment was conducted at early stages of the plants' phenological development, when the leaf tissues and meristem were most sensitive to herbivore infestation and fungal diseases. The plants were treated three times: first in phase BBCH 22 (winter wheat), BBCH 12 (maize), and BBCH 21 (winter oilseed rape); second in phase BBCH 40 (winter wheat), BBCH 18 (maize), and BBCH 50 (winter oilseed rape); and third in phase BBCH 73 (winter wheat), BBCH 39 (maize), and BBCH 73 (winter oilseed rape).
In each version of the experiment, damage caused by major insect pests was assessed: damage to oilseed rape siliques caused by cabbage seed weevils, damage to wheat leaves caused by cereal leaf beetles, and damage to maize plants caused by European corn borers. Moreover, the percentage of infestation with fungal diseases and the yield of the crops were assessed.
The experiments were carried out using the methods described in Zamojska et al. [14].

Assessment methods
Plants, leaves, and siliques were always selected randomly within each plot.
The siliques were collected from the highest oilseed rape sprouts, considering the fact that the highest sprouts are the ones most heavily attacked by cabbage seed weevils. One rape silique, regardless of its size, was randomly selected from each plant.
Hundred plants of winter wheat were randomly collected from each plot. One stem was then collected from each plant.

Assessment of fungal infestation
The assessments were carried out using the methods described in Zamojska et al. [14]. This involved the following: -Winter oilseed rape: gray mold (causal agent: Botrytis cinerea Pers) and Alternaria disease (causal agent: Alternaria spp.

Statistical analysis
The results were analyzed statistically with ARM Revision 2018 software. The assumption of the homogenity of variance attests that the data have been derived from normal distributions with an equal variance. The normality of the distributions for the studied traits was tested using Shapiro-Wilk's normality test. The Levene test is used to test the hypothesis that the variances across groups are equal. The two-way mixed analysis of variance (ANOVA) was carried out to determine the effects of years and fertilizers as well as years-fertilizers interaction on the variability of examined traits, for each trait independently. The mean values and standard deviations of traits were calculated. The Fisher's least significant differences (LSDs) were calculated for individual traits, and on this basis homogeneous groups were determined.
Ethical approval: The conducted research is not related to either human or animal use.

ANOVA results indicated that the main effects of year and
year × fertilizer interaction were significant for all the traits of study (P < 0.001). The results of the experiments on winter oilseed rape (  Table 3). The flag (F 2;18 = 666,500; P < 0.001) and subflag leaves (F 2;18 = 334,800; P < 0.001) of the plants fertilized with Ti were less damaged by cereal leaf beetles (Oulema melanopus L.), especially in 2014. The infestation  In all cases, there were statistically significant differences (P < 0.001) in the occurrence of pests and pathogens between the control version and the versions with Ti. Both in 2014 and 2015, the yields of winter oilseed .00 a ± 0.08 7.00 b ± 0.05 4.00 c ± 0.03 Foot rot 12.00 a ± 0.03 3.00 b ± 0.03 0.00 c ± 0.00 Sooty mold 20.00 a ± 0.05 1.00 b ± 0.02 1.00 b ± 0.02 Glume blotch 10.00 a ± 0.04 1.00 b ± 0.05 1.00 b ± 0.04 Fusarium head blight 10.00 a ± 0. .00 a ± 0.03 0.00 c ± 0.00 5.00 b ± 0.03 Sooty mold 15.00 a ± 0.03 0.00 b ± 0.00 0.00 b ± 0.00 Glume blotch 5.00 a ± 0.07 1.00 b ± ±0.03 1.00 b ± 0.04 Fusarium head blight 10.00 a ± 0.08 0.00 b ± 0.00 0.00 b ± 0.00 Yield (tonnes/ha) 8.13 c ± 0.02 8.35 b ± 0.02 8.49 a ± 0.02 The same superscript letters in each row denote a lack of significant differences. Percentage of plants infested with fungi Fusarium stalk rot 4.00 a ± 0.14 0.80 b ± 0.03 0.00 c ± 0.00 Maize smut 5.00 a ± 0.14 0.00 b ± 0.00 0.00 b ± 0.00 Brown spot 0.00 a ± 0.00 0.00 a ± 0.00 0.00 a ± 0.00 Yield (tonnes/ha) 8.57 c ± 0.04 9.83 a ± 0.04 9.31 b ± 0.04 The same superscript letters in each row denote a lack of significant differences. rape, winter wheat, and maize obtained from the Ti versions were always significantly higher than the yield from the control samples. A higher dose of Ti was more effective for the majority of parameters.

Discussion and conclusions
The experiments proved the positive effect of the preparation containing Ti ions, which limited the effects of biotic stresses. The preparation reduced damage caused by pests and the infestation of plants with fungal diseases. The fertilizer reduced damage to winter rapeseed and winter wheat plants caused by their major pests. All the plants were less infested with fungal diseases and their yield increased. The direct factor explaining this phenomenon is unknown because the available scientific publications do not provide an answer to it. It seems that the plants in which stress was reduced with Ti compounds were more resistant to fungal diseases and harmful insects. The fact that Ti has many positive effects on plants might be an indirect cause of this phenomenon. For a long time, Ti has been known to increase the yield of crops. Its beneficial effect has been described in scientific reports [3][4][5]. However, researchers have not explained this effect in detail. One of the major properties of the Tytanit preparation is the fact that it increases the uptake of nutrients by the root system, especially iron and potassium [7]. Plants may have proteins that (either specifically or nonspecifically) bind with Ti. Plants treated with Ti might be strengthened because the preparation stimulates physiological processes in them. In consequence, plants are in a better condition. They are less susceptible to diseases and less attractive to pests. This relationship between the extent of damage caused by pests and the degree of infestation with fungal diseases has been described in scientific articles [13][14][15].
It is also known that any environmental stress (biotic or abiotic) has an influence on metabolic changes in plants. Ti may participate in plants' defensive processes, which may cause changes in tissues and inhibit the growth and development of pests' eggs and larvae [16]. There have been studies providing evidence that the quality of plant tissues has an influence on the survival of insects and that there are interactions between the larval growth rate and population size and the plant tissue structure [17]. Some herbivorous insects have chemoreceptors to sense the tissue quality before they attack. They can identify the thickness of plant tissues and sense the emission of substances such as ethylene. It is likely that Ti also influences volatile and visual features (which may be important for the selection of the host).
There have been numerous scientific reports describing the positive effects of Ti applied to plants' roots or leaves; for example, the increase in the chlorophyll content and photosynthesis intensity in rape plants, a heavier 1,000 kernel weight of wheat, higher sugar content in sugar beets, the reduced effect of Curvularia leaf spot, and the lower incidence of bacterial leaf blight in maize [18]. Bacilieri et al. [5] observed that TiO 2 nanoparticles increased the activity of superoxide dismutase, catalase, and glutathione peroxidase in maize at the four-leaf stage. Lu et al. [19] proved that TiO 2 increased the absorption of water and nutrients by soy plants, which strengthened their antioxidative capacity and accelerated their development. Other examples of the positive effect of Ti are increased biomass production in bell peppers (Capsicum annuum L.) [20], increased plant height and a greater number of leaves in snapdragons (Antirrhinum majus L.) [21], increased plant growth and quality in zonal geraniums (Pelargonium hortorum L.) [21], increased seed yield, 1,000 kernel weight, and seed germination in Timothy grass (Phleum pretense L.) [22], and increased yield, better fruit quality, higher vitamin content, and higher macronutrient uptake in tomatoes (Solanum lycopersicum L.) [7]. Other authors proved that the foliar application of Ti significantly affected the activity of antioxidative enzymes (superoxide dismutase, catalase, and glutathione peroxidise), the content of malondialdehyde, and protein in maize plants [23]. According to Kovacik et al. [24], the Tytanit fertilizer positively influenced the formation of the aerial and underground phytomass of winter oilseed rape. Ercoli et al. [25] also observed that the application of Ti resulted in a higher yield of maize seeds. There have also been reports on the beneficial effect of Ti, which reduced the incidence of diseases and increased the yield of cowpeas (Vigna unguiculata Walp.) [6].
This research proves that the inclusion of treatments with preparations containing Ti ions into the technology of production of the plant species being studied increased the effectiveness of integrated crop protection programs. In view of the increasing problem of pests' resistance to crop protection products, the essential rule of the prevention strategy assumes a reduction in the selection pressure of insecticides and fungicides. The results of the experiment show that it is possible to achieve this goal by limiting the occurrence of pests and diseases by means of stimulants containing absorbable Ti. The research findings should be used in strategies for preventing pests' and pathogens' resistance and as the basis for creating integrated crop protection programs. The research will be continued in other field crops. Conflict of interest: The authors declare no conflict of interest.
Data availability statement: All data generated or analyzed during this study are included in this published article.