In the last two decades, the horse chestnut (Aesculus hippocastanum L.), introduced into the steppe zone of Ukraine, has been severely affected by the horse chestnut leaf miner Camereraria ohridella Deschka & Dimič, 1986, which results in damage to the assimilating organs, premature leaf defoliation and, as a consequence, a significant reduction in the reserve substances required for normal life of the plant. In recent studies, the main focus has been placed on the study of the pest’s effects on the non-enzymatic antioxidant protection system of the representatives of the genus Aesculus, while the enzymatic system of horse chestnut protection from the active forms of oxygen under stress is still poorly understood. The purpose of this study was to evaluate the reaction of catalase and two peroxidases of A. hippocastanum leaves, which differ in the level of damage by C. ohridella. The intensity of damage to A. hippocastanum leaves by the horse chestnut leaf miner in the park zones and botanical gardens of Dnipro city was determined, the activity and isoenzyme composition of benzidine-peroxidase, activity of guaiacol-peroxidase and catalase were measured. The lowest average benzidine-peroxidase activity was found in the group of trees with low level of leaf blight and the highest activity – in the group with high level. The opposite dependence was shown by catalase, the activity of which significantly decreases with increasing level of damage inflicted by the phytophage on the chestnut’s assimilating organs. Based on the determination of the variation coefficients, it has been shown that benzidine-peroxidase activity has a higher level of variability than that of catalase and guaiacol-peroxidase. It is established that under the influence of the leaf miner, activity of guaiacol-peroxidase was significantly higher by 87.1% and 75.6%, respectively, for medium and high levels of damage caused to the leaf by this phytophage as compared to that for low levels of damage. The increased level of leaf damage caused by the phytophage is reflected in the change in the isozyme profile of benzidine-peroxidase. The high activity of benzidine-peroxidase in the leaves of A. hippocastanum is due to the presence of several molecular forms that exhibit maximum activity in the narrow pH range (4.15–4.69). Quantitative redistribution of activity between the different molecular forms of benzidine peroxidase can be considered as the main regularity of changes in the expression of benzidine-peroxidase caused by different levels of leaf damage. The results showed that only one benzidine-peroxidase isoform with an isoelectric point of 4.15 shows a significant increase in activity (on average by 2.1 times) in A. hippocastanum leaves with medium and high levels of damage by C. ohridella. Significant reduction in activity is reported for dominant isoperoxidase with an isoelectric point of 4.25 revealing medium pest damage, and for high damage only a decreasing tendency is shown. The data obtained show that horse chestnut trees can specifically respond to mechanical damage by C. ohridella to leaves due to the changes in the activity of individual molecular forms of peroxidase. Further studies of oxidative metabolism are needed to understand the formation of resistance of representatives of the Aesculus genus to damage caused by this moth species based on a wider range of redox enzymes.

This study analyzed the dynamics of leaf damage, the content of easily soluble proteins, benzidine-peroxidase activity and the composition of acid enzyme isoforms in the leaves of Aesculus hippocastanum L. under the influence of the chestnut miner Cameraria ohridella Deschka et Dimić (Lepidoptera, Gracillariidae) in plantations in one of the Ukraine’s largest industrial cities Dnipro, located in the steppe zone of Ukraine. During July-August, the destruction of leaves by the pest in relatively stable horse chestnut trees increased to 13.3%, while in unstable trees it increased to 97.5%. The maximum amount of protein was found in horse chestnut leaves in July, which decreased significantly in August and correlated with the level of leaf damage by the pest. The horse chestnut leaves were characterized by a more than twofold increase in cytoplasmic peroxidase activity in cases of high level of damage to the leaves by the phytophage. These observations indicate that consumption of the leaves by C. ohridella causes oxidative stress, which leads to the activation of enzyme. The horse chestnut trees have been shown to respond specifically to the attack of C. ohridella due to changes in the activity of individual peroxidase isozymes, the expression of which varies across the spectrum. In the leaves of the horse chestnut trees with a high level of damage by miner, the expression of enzyme isoforms in the pH range of 4.08–4.15 is significantly activated, which can be considered as a reliable biochemical marker of plant sensitivity of Ae. hippocastanum to phytophagous attack by C. ohridella. However, increase in the degree of phytophage invasion does not change the activity of isoperoxidase 4.21, and isoperoxidase with pI 4.25, 4.42 and 4.58 correspond to a decrease in activity compared with relatively resistant Ae. hippocastanum plants. We noted that the high total activity of peroxidase, as well as active adjustments in the spectrum of the peroxidase system, obviously, contribute to maintaining the functional integrity of the photosynthetic system of leaves of Ae. hippocastanum under the influence of the chestnut miner C. ohridella by neutralizing reactive oxygen species.

The paper presents the results on resistance of Aesculus hippocastanum Linnaeus, 1753 trees to Cameraria ohridella Deschka & Dimić, 1986 (Lepidoptera, Gracillariidae) impact under conditions of a modern urban environment on the example of Dnipro city as the largest industrial city in Ukraine. Field experiments were conducted in all park areas of the city, which allowed covering the full gradient of the existing urban environment and considered the different degrees of the tree settlement by the invasive insect species. The research of the impact of C. ohridella caterpillars’ vital activity on the photosynthetic apparatus state was carried out by applying a chlorophyll fluorescence induction technique. Diagnosis of photosynthetic dysfunction of fresh Ae. hippocastanum leaves was conducted using a portable “Floratest” fluorometer manufactured in Ukraine. Interpretation of the obtained Kautsky curves showed that significant changes in their critical parameters associated with the degree of leaf damage by C. ohridella caterpillars were not detected. The influence of tree growth site conditions on the following 4 main indicators of chlorophyll fluorescence induction was established: the initial value of fluorescence induction after irradiation; the value of “plateau” fluorescence induction; the maximum value of fluorescence induction; the stationary value of fluorescence induction after light adaptation of the plant leaf. It was found that the efficiency coefficients of photochemical processes in Ae. hippocastanum trees growing in low terrain levels differed significantly, which can probably be interpreted as their response to the specific characteristics of the urban environment.

The complex of invasive species of phytophagous insects that can adapt to novel living conditions is constantly increasing. The ecological effect of their vital activity in the new environment is almost impossible to predict. As a result, invasions undesirable in the economic sense are often observed. The horse -chestnut leaf miner (Cameraria ohridella Deschka & Dimič, 1986, Gracillariidae) is one of these problematic invader species in the introduced range of the horse chestnut (Aesculus hippocastanum (Linnaeus, 1753), Sapindaceae). We studied the effect of C. ohridella on the state of the photosynthetic apparatus in Ae. hippocastanum leaves. Photosynthesis is the one of the processes most vulnerable to stress factors, so information about the state of photosynthetic apparatus in a plant under the influence of phytophage feeding obtained with fluorescence analysis can be significant. The feeding effect of C. ohridella caterpillars on the functional state and activity of Ae. hippocastanum photosynthetic apparatus was studied. We studied critical parameters of chlorophyll fluorescence induction reflecting the effect of a single C. ohridella generation (feeding the caterpillar for the five stages of this species’ development) on the functional links of the photosystem II in Ae. hippocastanum leaves. The data obtained show a decrease in PS II quantum efficiency (inhibition of photosynthetic activity) in the leaves from different parts of the crown; it suggests the destructive effect of C. ohridella caterpillar feeding on Ae. hippocastanum photosynthetic apparatus. Values of all key parameters of chlorophyll fluorescence indication evidence inhibition of photophysical and photochemical processes of photosynthesis and impaired coherence of Calvin cycle reactions. Study of the feeding effect of C. ohridella caterpillars on the efficiency of the main enzyme in the Calvin cycle (which closely correlates with the coefficient of fluorescence induction that characterizes the efficiency of dark photosynthetic processes) showed a significant decrease in its activity in the leaves of both the illuminated and shaded parts of the crown. Our study has shown that the method of chlorophyll fluorescence induction allows one to determine the general state of a plant in an express regime by evaluating the main process of plant life as photosynthesis. Analysis of chlorophyll fluorescence parameters is a powerful and effective tool for determining the effect of phytophages on the plant body. The obtained data allow us to apply the method of analyzing chlorophyll fluorescence induction in practice to establish the physiological state of tree flora in forests and garden farms.

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