PEER-REVIEW ARTICLE PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9126 Biotic Stress Responses and Oxidative Defense Mechanisms of Pinus brutia against Pine Processionary Moth Infestations Ergin Yilmaz ,a,* Esra Nurten Yer Çelik ,b Orhan Gülseven ,c Şeyma Selin Akin ,d Nezahat Turfan ,e and Sezgin Ayan ,b * Corresponding author: yilmazergin@kastamonu.edu.tr DOI: 10.15376/biores.20.4.9127-9147 GRAPHICAL ABSTRACT https://orcid.org/0000-0003-1471-3741 https://orcid.org/0000-0002-6368-3916 https://orcid.org/0000-0001-9242-8394 https://orcid.org/0000-0001-8353-6422 https://orcid.org/0000-0002-5753-0390 https://orcid.org/0000-0001-8077-0512 PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9127 Biotic Stress Responses and Oxidative Defense Mechanisms of Pinus brutia against Pine Processionary Moth Infestations Ergin Yilmaz ,a,* Esra Nurten Yer Çelik ,b Orhan Gülseven ,c Şeyma Selin Akin ,d Nezahat Turfan ,e and Sezgin Ayan ,b Defense mechanisms were studied for Pinus brutia, a cornerstone Turkish forest tree, against pine processionary moth damage by Thaumetopoea pityocampa (Den. & Schiff.) and Thaumetopoea wilkinsoni Tams 1926 moth species. This research addressed the significance of Pinus brutia in afforestation and breeding. The expression of enzymatic antioxidants (SOD, POD, CAT, APX) and photosynthetic pigments (chlorophylls and carotenoids) at a clonal level in response to insect damage was assessed. Approximately 84 needle samples from 28 Pinus brutia clones from the Antalya Düzlerçamı Brutian Pine Seed Orchard were studied. Samples were collected in February and August 2021 to capture responses during key insect activity periods. These samples were then analyzed for pigment concentrations and antioxidant activities. Statistical analysis revealed that sampling period and clone significantly affected chlorophyll and carotenoid levels. The POD and SOD activities were primarily influenced by the sampling period. However, CAT activity was affected by the number of insect pouches, the period, and the clone. APX activity was significantly impacted by both pouch number and sampling period. These findings offer insights into how seasonal changes and genetic variations modulate P. brutia clones' defense mechanisms against pine processionary moth infestations, informing future forest management. DOI: 10.15376/biores.20.4.9127-9147 Keywords: Pinus brutia; Enzymatic antioxidants; Photosynthetic pigments; Clonal variation; Oxidative defense; Biotic stress; Pine processionary moth Contact information: a: Kastamonu University, Vocational School, Department of Pharmacy Services, Kastamonu, Turkiye; b: Kastamonu University, Faculty of Forestry, Department of Silviculture, Kastamonu, Turkiye; c: Kastamonu University, Institute of Science, Kastamonu, Turkiye; d: Kastamonu University, Institute of Science, Kastamonu, Turkiye; e: Kastamonu University, Faculty of Science and Literature, Biology Department, Kastamonu, Turkiye; * Corresponding author: yilmazergin@kastamonu.edu.tr INTRODUCTION Pinus brutia Ten. is a primary forest tree species with a natural distribution in the Mediterranean and Aegean regions of Turkiye and the Eastern Aegean Islands; its wide areal range reflects high adaptation to Mediterranean climatic zones (Quezel 1979). The natural range of the species includes Crete, Cyprus, Syria, and northern Iraq, and in recent years it has been introduced into several countries with Mediterranean climates (Selik 1958; Critchfield and Little 1966; Arbez 1974; Panetsos 1981; Kara et al. 1997). It is tolerant of drought (Oppenheimer 1967; Nahal 1983) and is able to grow on different soil types (Quézel 1985, 2000; Milios et al. 2019). Pinus brutia is an important species for https://orcid.org/0000-0003-1471-3741 https://orcid.org/0000-0002-6368-3916 https://orcid.org/0000-0001-9242-8394 https://orcid.org/0000-0001-8353-6422 https://orcid.org/0000-0002-5753-0390 https://orcid.org/0000-0001-8077-0512 PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9128 rehabilitating degraded lands in the Mediterranean basin. As an endemic species native to the eastern Mediterranean region (Kaya and Raynal 2001), it is preferred in afforestation and reclamation efforts in Turkiye because of its rapid growth (DPT 2001). It stands out as a commercially important forest species (Usta 1990; Fady et al. 2003; Michelozzi et al. 2008). Forest ecosystems are complex networks of interactions between trees, plants, animals, and microorganisms. Important factors threatening these ecosystems’ integrity are insects and the herbivory damage that they cause (Avcı 2000). Thaumetopoea wilkinsoni (common in Turkiye and the Middle East) and Thaumetopoea pityocampa (common in Europe and North Africa) are among the most important defoliators of Pinus species in the Mediterranean Basin (Denis and Schiffermüller 1776; Masutti and Battisti 1990; Vega et al. 1997; Carus 2004; Rodríguez-Mahillo et al. 2012). The pine processionary moth is a widespread phytophagous species both globally and in Anatolia. It consumes the needles of Pinus species, an important component of Anatolian forests, leading to a decrease in the growth rates of trees (Kanat et al. 2005; Durkaya et al. 2009). It is widely distributed in warm regions of Anatolia under the influence of Mediterranean climate (Çanakçıoğlu 1993; Kanat and Türk 2002). This species, which causes significant economic losses in forest areas, can cause annual growth losses of up to 60% in Pinus brutia, Pinus nigra, and other Pinus species (Anonymous 1995). Thaumetopoea spp. larvae cause damage by feeding on the needles of Pinus species. While at low population densities they usually damage the twigs around their sacs, at epidemic levels they can cause defoliation and even desiccation of the trees. At later stages of larval development, the severity of damage increases in parallel with increasing nutrient requirements, reaching a maximum in the last instar larvae (Devkota and Schmidt 1990). The annual life cycle of pine processionary moth-induced defoliation negatively affects the long-term health of Pinus forests. Reduced annual growth of infected trees leads to physiological weakening and thus increased vulnerability to other biotic (secondary pests, pathogens) and abiotic (drought, temperature stress) stressors (Myteberi et al. 2013). Insect-induced herbivory triggers several biochemical processes in plant tissues that disrupt cellular homeostasis. One of these processes is the rapid and transient increase of reactive oxygen species (ROS) such as superoxide anion O2 .- and hydrogen peroxide (H2O2). This ROS production represents one of the early defense responses of plant cells against damage. Increased ROS levels induce activation of the enzymatic antioxidant system, which plays an important role in plant metabolism. Superoxide dismutase (SOD) is a metalloenzyme that dismutates O2 -1 into H2O2 and molecular oxygen (O2). Peroxidases (POD) detoxify H2O2 by oxidizing phenolic compounds (Skwarek et al. 2017). PODs are critical to plants’ rapid defense mechanisms against insect damage (Gulsen et al. 2010; Usha Rani and Jyothsna 2010). Catalase (CAT), which has a central role in combating oxidative stress, is one of the first antioxidant enzymes discovered. The CAT catalytically cleaves H2O2 into water (H2O) and O2, thereby eliminating its toxic effect (Kerchev et al. 2016). The localization of CAT enzyme in different cellular compartments (mitochondria, thylakoid, and stroma of chloroplasts, cytosol and peroxisomes) and its high affinity for H2O2 enable it to function as an effective H2O2 scavenger in stressed plants and consequently play an important role in preventing cellular damage (Mushtaq et al. 2020). In plants, oxidative status constitutes a fundamental element of defense mechanisms against various stress factors. Rapid and transient reactive oxygen species (ROS) production is observed as a common physiological response under biotic and abiotic stress conditions (Maffei et al. 2007; Torres 2010). ROS, bifunctional molecules, play a role in signal transduction processes and can cause toxic effects at high PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9129 concentrations. Biotic stress-induced ROS production mechanisms and their physiological importance are among the current research topics (Maffei et al. 2007). The sudden and significant increase in ROS levels under stress conditions is defined as “oxidative burst” (Hare et al. 2011). Increases in ROS production have been found in peroxisomes, mitochondria and plasma membranes following herbivore insect damage (Maffei et al. 2007; Torres 2010). This ROS burst may constitute an early phase of induced defense mechanisms against pathogens and herbivores, acting as a protective barrier against subsequent attacks (Powell et al. 2006). Due to their high reactivity, ROS can cause oxidative damage by interacting with essential biomolecules such as proteins, lipids, and nucleic acids. To prevent this potential auto-toxicity, plant cells have evolved antioxidant defense systems that remove excess ROS and maintain ROS concentration at low and stable levels (Maffei et al. 2007; Howe and Jander 2008). Temperature increases observed worldwide due to global climate change are causing a significant increase in Thaumetopoea wilkinsoni and Thaumetopoea pityocampa population densities. This increases the extent of herbivory damage to Pinus species (Leblebici et al. 2023). Considering the ecological and economic importance of Pinus forests worldwide and in Turkiye, it is of great importance to investigate in detail the damage caused by these defoliator species and the effects of biotic stress induced by them on oxidative stress. Pinus brutia Ten. is one of Turkiye's important forest tree species, and breeding studies have significantly progressed. In this context, there is a need to determine different clones’ resistance or sensitivity levels against pine processionary moth (T. pityocampa and T. wilkinsoni) damage. This study considered the seasonal variations of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids) and enzymatic antioxidants (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)) to determine the resistance or susceptibility of different clones in P. brutia, where pine processionary moth damage was intensively observed. In this study, the resistance levels or sensitivities of Pinus brutia clones to pine processionary moth were evaluated. The study examined changes in the photosynthetic pigments and antioxidant enzyme levels to reveal the biological defenses of different clones against pine processionary moth and their resistance to oxidative stress. In this context, the biological responses of clones to pine processionary moth and the relationship between these responses and resistance were investigated. The basic hypotheses in the study are as follows. Pinus brutia clones exhibit varying levels of resistance or susceptibility to herbivore damage by Thaumetopoea species, depending on genotypic differences. Thaumetopoea damage triggers an oxidative stress response in Pinus brutia clones and causes a significant seasonal or interclonal effect on enzymes (SOD, POD, CAT, APX). This approach and hypotheses enabled collecting more detailed clone-based data related to pine processionary moth, which is critically important for forest management and breeding studies. MATERIALS AND METHODS Materials The vegetative material of this research was obtained from the clonal seed orchard of Gölhisar provenances (Pinus brutia Ten.). The Brutian pine with national registration number 8, was planted in 1980 and located within the borders of Antalya Forest PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9130 Management Directorate Düzler Pine Chiefdom. This seed orchard was established with 28 different clones representing different genotypes. Within the scope of this study, needle leaf samples were collected from three genetic replicates (ramet) of each clone, recording the number of pines processionary moth pouches on the trees. Sampling was carried out during two different phenological periods in 2021: February (Period I), the dormancy period when vegetation has not started, and August (Period II), the active growth phase. The needle samples from three ramet of each clone were transferred to the Central Research Laboratory of Kastamonu University and stored at -80 °C until biochemical analyses. Methods All samples were collected from the uppermost lower branches of the trees’ southern sides, which could be reached with pruning shears. The southern side represents an area where harmful populations may be concentrated because it receives more sunlight. Samples were collected from pine needles during two distinct periods when damage from the pine processionary moth was either high or low. The dependent variables examined in this study were photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids) and enzymatic antioxidants (SOD, POD, CAT, and APX). To extract and quantify photosynthetic pigments, 0.5 g of fresh needle leaf samples were taken and frozen in liquid nitrogen and powdered. The powdered samples were extracted using 10 mL of 80% acetone solution. After homogenization, the suspension was centrifuged at 3000 rpm for 10 minutes. It was centrifuged at (+4 °C). 3 mL of supernatant was used. Following centrifugation, the clear supernatant was taken and determinations were made for the amounts of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids in it, spectrophotometrically (Shimadzu brand, UV Pharmaspec 1700 model, Kyoto-Japan). Absorbance values, recorded as A (absorbance), represent a measure of how much light is absorbed by the substance at specific wavelengths using a spectrophotometer. Absorbance values were read in a spectrophotometer at wavelengths of 450 nm (carotenoids), 645 nm (chlorophyll b), and 663 nm (chlorophyll a), respectively. Total chlorophyll concentration was calculated using the equation described by Arnon (1949). Total carotenoid concentration was determined using a modified version of the Jaspars formula (Witham et al. 1971), Chl a = [12.7 (A663) - 2.69 (A645)] (V/1000×W) (1) Chl b = [22.9 (A645) - 4.68(A663)] (V/1000×W) (2) Total chl a+chl b = [20.2 (A645) + 8.02 (A663)] (V/1000xW) (3) Total carotenoid = (4.07 × A450) – (0.0435 × chl a amount + 0.367 × chl b amount) (4) where V is a volume of 80% acetone, and W is wet weight (g) of the extracted leaf sample. In order to determine the enzymatic antioxidant activities in the samples, 0.5 g of fresh needle leaf samples were flash frozen in liquid nitrogen and powdered. Then the obtained powder material was homogenized with 5 mL of cold extraction buffer containing 0.1 M potassium phosphate buffer (KH₂PO₄). The pH value was studied as 7. The homogenate was centrifuged at 15000 rpm for 15 min at +4 °C and obtained the supernatant. Enzyme activities were analyzed in this supernatant by spectrophotometric methods. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9131 Catalase (CAT) activity was determined spectrophotometrically according to the protocol modified by Gong et al. (2001). This method monitored the rate of breakdown of hydrogen peroxide (H₂O₂) at a wavelength of 240 nm. Superoxide dismutase (SOD) enzyme activity was determined by spectrophotometric method based on the principle of nitroblue tetrazolium (NBT) reduction inhibition applied by Agarwal and Pandey (2004). The SOD activity was calculated by measuring the amount of enzyme inhibiting NBT reduction of superoxide radicals in the reaction mixture. Peroxidase (POD) activity was determined by the spectrophotometric method described by Yee et al. (2002). In this method, the increase in absorbance of the colored product formed by the oxidation of guaiacol by POD in the presence of hydrogen peroxide was monitored at 470 nm wavelength. Ascorbate peroxidase (APX) activity was determined spectrophotometrically according to the method developed by Nakano and Asada (1981). In this method, the extent of absorbance decrease caused by the oxidation of ascorbate to dehydroascorbate by APX in the presence of hydrogen peroxide was measured at 290 nm wavelength. Statistical Evaluation The relationships between dependent variables (chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, SOD, POD, CAT and APX activities) obtained from Pinus brutia needle leaf samples and independent variables (number of pouches, sampling period, clone and number of pouches × clone interaction) were examined by linear regression analysis using R statistical software. Analysis of Variance (ANOVA) was applied to determine the main and interaction effects of independent factors (clone, number of pouches, period and clone × number of pouches) on the variables analyzed. Duncan Multiple Comparison Test was used to determine homogeneous groups and to make multiple comparisons between means in variables showing significant differences according to ANOVA results. Significance level was accepted as P < 0.05 in statistical analyses. RESULTS AND DISCUSSION The results of statistical analysis between enzymatic antioxidant activities (SOD, POD, CAT, APX) and independent variables (number of pouches, sampling period, clone and pouch number × clone interaction) are presented in Table 1. The data presented in Table 1 show that enzymatic antioxidant activities (SOD, POD, CAT, APX) were highest in February, when intense biotic stress from pine processionary moth (Thaumetopoea spp.) damage was observed. However, these activities decreased significantly in August, when damage decreased. This finding suggests that plants combat oxidative stress by activating their enzymatic antioxidant systems against pine processionary moth attack, and that these defense mechanisms revert to their previous state when the stress load decreases. Similarly, the results of statistical analysis between photosynthetic pigment concentrations (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids) and the same independent variables are summarized in Table 2. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9132 Table 1. Evaluation of Enzymatic Antioxidant Activities by Linear Regression Analysis Dependent Variable Constant Value (β0) Independent Variable Number of Pouches Period Clone F P Forecast P Forecast P F P APX (EU mg/protein) 390.6503 <0.0001 0.0019703 0.0121 -0.016797 0.0043 1.2135 ns CAT (EU mg/protein) 1158.4894 <0.0001 0.00703 0.0067 -0.08553 0.0001 1.8086 0.0315 POD (EU mg/protein) 90.8301 <0.0001 -0.011845 ns -0.75062 0.0001 0.8884 ns SOD (EU mg/protein) 69.43603 <0.0001 -3.266 ns -73.925 0.0001 0.89163 ns The seasonal effect of pine processionary moth damage on photosynthetic pigments is a significant finding. Chlorophyll a, b, and total chlorophyll amounts decreased across the sampling period, but the amount of chlorophyll and interactions among clones had limited effects on the pigments. Table 2. Evaluation of Photosynthetic Pigment Levels with Linear Regression Equations Dependent Variable Constant Value (β0) Independent Variable Period Clone F P Forecast P F P Chlorophyll a (mg/g) 5934.797 <0.0001 -0.016080 0.0001 2.252 0.0052 Chlorophyll b (mg/g) 2715.977 <0.0001 -0.0164283 0.0001 1.3667 ns Total Chlorophyll (mg/g) 5181.577 <0.0001 -0.03248 0.0001 1.957 0.0172 Carotenoid (mg/g) 6111.8 <0.0001 -0.77481 0.0001 1.684 0.0503 The effects of independent variables (number of pouches, sampling period, clone and number of pouches × clone interaction) on APX, CAT, POD, and SOD enzyme activities were analyzed. According to the results of linear regression analysis, the significant effects of pouch number and sampling period on APX activity were determined (P < 0.05). In contrast, the effects of clone and pouch number × clone interaction were not statistically significant (P > 0.05). While APX activity levels decreased from February to August, increased APX activity was observed with increased pouches. CAT activity was significantly affected by the number of pouches, sampling period and clone factors (P < 0.05), but the effect of pouch number × clone interaction was not significant (P > 0.05). CAT activity also tended to decrease periodically, while an increase in CAT activity was detected with the increase in pouches. For POD activity, the sampling period factor was found to be significant (P < 0.05); the effect of other factors was not statistically significant (P > 0.05). The POD activity levels decreased with the transition from February to August. Similarly, only the sampling period had a significant effect on SOD activity (P < 0.05), while the effect of other factors was not statistically significant (P > 0.05). The SOD activity levels also showed a periodic decrease from February to August. According to the results of analysis of variance (ANOVA) and Duncan's Multiple Comparison Test, we present the homogeneous groups of sampling periods (February and August) for SOD, POD, CAT, and APX enzyme activities in Table 3. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9133 Table 3. Variation of Enzymatic Antioxidant (SOD, POD, CAT, APX) Activities in Different Pinus brutia Clones in I- (February) and II- (August) Periods PERIOD I PERIOD II SOD Enzyme Activity (EU/mg protein) Clone No. Pouches No. X±Sx Groups Clone No. Pouches No. X±Sx Groups 8586 7 43,85±1,36 d 8570 8 19,12±2,20 f 8582 4 44,23±13,01 d 8571 8 21,46±1,41 ef 8562 8 45,03±5,23 d 8569 10 22,66±3,27 def 8573 8 48,43±6,54 d 8574 4 25,36±2,74 def 8572 3 52,64±3,13 d 8581 4 28,96±3,40 def 8583 6 53,82±8,30 d 8585 4 29,42±5,46 def 8581 4 54,46±8,17 d 8575 4 33,62±4,88 def 8569 10 55,84±12,12 d 8580 5 34,67±3,44 def 8579 4 59,87±4,20 d 8578 6 37,94±5,09 cdef 8563 4 61,84±8,36 d 8587 5 38,67±4,20 cdef 8565 3 63,87±12,45 d 8561 6 38,82±10,45 cdef 8564 5 78,47±15,85 d 8565 3 40,96±4,99 cdef 8570 8 81,44±11,43 d 8562 8 41,70±8,29 cdef 8571 8 82,81±5,88 d 8563 4 42,22±6,47 bcdef 8580 5 84,35±15,14 d 8583 6 42,90±12,01 bcdef 8578 6 92,45±9,57 d 8566 3 43,15±6,01 bcdef 8587 5 104,22±11,35 cd 8576 3 46,75±3,77 bcdef 8576 3 115,68±21,78 cd 8586 7 51,70±4,51 bcdef 8567 4 117,40±25,05 cd 8577 6 51,81±8,11 bcdef 8577 6 117,70±18,39 cd 8564 5 54,00±13,98 bcdef 8560 4 123,22±32,28 cd 8579 4 54,48±6,55 bcdef 8584 4 123,61±9,25 cd 8567 4 54,78±5,68 bcdef 8561 6 170,77±67,72 cd 8582 4 58,12±19,74 bcdef 8574 4 179,51±38,48 cd 8584 4 59,27±5,58 bcde 8575 4 186,36±69,22 cd 8560 4 61,67±9,46 bcd 8568 4 270,18±79,24 b 8572 3 75,71±21,16 abc 8566 3 407,71±170,49 ab 8568 4 80,82±11,20 ab 8585 4 444,63±162,07 a 8573 8 103,72±40,63 a F-value P-level 3,758 0,000 F-value P-level 2,724 0,000 PERIOD I PERIOD II POD Enzyme Activity (EU/mg protein) Clone No. Pouches No. X±Sx Groups Clone No. Pouches No. X±Sx Groups 8586 7 0,16±0,01 h 8563 4 0,001± 0,0003 h 8580 5 0,21±0,01 gh 8580 5 0,001± 0,0001 h 8583 6 0,21±0,01 gh 8583 6 0,002± 0,0009 gh 8572 3 0,23±0,04 gh 8578 6 0,003± 0,0005 gh 8562 8 0,23±0,03 gh 8577 6 0,003± 0,0005 gh 8569 10 0,29±0,04 fgh 8571 8 0,003± 0,0008 fgh 8565 3 0,33±0,06 efgh 8562 8 0,003± 0,0002 fgh 8571 8 0,48±0,13 defgh 8575 4 0,004± 0,0009 efgh 8579 4 0,52±0,15 cdefgh 8569 10 0,004± 0,0005 defgh 8581 4 0,59±0,19 cdefgh 8568 4 0,004± 0,0012 defgh 8564 5 0,61±0,08 cdefgh 8587 5 0,005± 0,0011 defgh 8573 8 0,66±0,09 cdefgh 8565 3 0,005± 0,0002 defgh 8561 6 0,69±0,17 bcdefgh 8584 4 0,005± 0,0011 8577 6 0,71±0,18 bcdefgh 8579 4 0,005± 0,0005 defgh 8576 3 0,74±0,16 bcdefgh 8570 8 0,005± 0,0006 defgh 8585 4 0,83±0,22 bcdefgh 8586 7 0,006± 0,0014 cdefg 8570 8 0,87±0,36 abcdefgh 8581 4 0,006± 0,0007 cdefg 8566 3 0,90±0,14 abcdefgh 8576 3 0,006± 0,0011 cdefg 8563 4 0,92±0,24 abcdefgh 8574 4 0,007± 0,0024 bcdefg PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9134 8578 6 0,93±0,21 abcdefgh 8585 4 0,008± 0,0021 abcdefg 8560 4 0,98±0,30 abcdefg 8582 4 0,009± 0,0017 abcdefg 8567 4 1,09±0,34 abcdef 8564 5 0,010± 0,0006 abcdef 8568 4 1,11±0,40 abcde 8566 3 0,011± 0,0037 abcde 8575 4 1,15±0,20 abcd 8561 6 0,011± 0,0031 abcd 8582 4 1,22±0,51 abcd 8560 4 0,012± 0,0020 abc 8584 4 1,31±0,27 abc 8572 3 0,013± 0,0033 ab 8574 4 1,48±0,06 ab 8573 8 0,015± 0,0056 a 8587 5 1,63±0,52 a 8567 4 0,015± 0,0047 a F-value P-level 2,997 0,000 F-value P-level 3,690 0,000 PERIOD I PERIOD II CAT Enzyme Activity (EU/mg protein) Clone No. Pouches No. X±Sx Groups Clone No. Pouches No. X±Sx Groups 8574 4 0,20±0,030 g 8560 4 0,17±0,014 e 8584 4 0,21±0,005 fg 8567 4 0,22±0,011 de 8587 5 0,21±0,024 fg 8565 3 0,23±0,010 cde 8568 4 0,22±0,057 fg 8577 6 0,23±0,018 cde 8566 3 0,22±0,036 fg 8586 7 0,23±0,019 cde 8578 6 0,24±0,019 fg 8566 3 0,24±0,005 cde 8575 4 0,28±0,034 efg 8576 3 0,25±0,020 cde 8560 4 0,31±0,038 defg 8572 3 0,26±0,072 bcde 8576 3 0,31±0,063 defg 8582 4 0,26±0,054 bcde 8585 4 0,32±0,028 defg 8584 4 0,27±0,050 bcde 8567 4 0,34±0,037 cdef 8578 6 0,27±0,041 bcde 8577 6 0,34±0,062 cdef 8564 5 0,28±0,021 bcde 8571 8 0,34±0,029 cdef 8580 5 0,28±0,014 bcde 8564 5 0,35±0,020 cdef 8583 6 0,29±0,021 bcde 8579 4 0,38±0,007 cde 8585 4 0,29±0,032 bcde 8583 6 0,40±0,030 cde 8570 8 0,30±0,035 bcde 8570 8 0,41±0,043 cde 8579 4 0,30±0,044 abcde 8561 6 0,41±0,040 cde 8581 4 0,31±0,024 abcde 8569 10 0,44±0,049 cd 8568 4 0,32±0,096 abcde 8562 8 0,44±0,064 cd 8563 4 0,32±0,004 abcde 8563 4 0,44±0,057 cd 8561 6 0,33±0,038 abcd 8573 8 0,45±0,030 cd 8562 8 0,34±0,028 abcd 8572 3 0,47±0,046 bc 8573 8 0,34±0,084 abcd 8565 3 0,48±0,017 bc 8574 4 0,38±0,029 abc 8582 4 0,60±0,098 ab 8587 5 0,38±0,086 abc 8586 7 0,61±0,017 a 8575 4 0,40±0,074 ab 8581 4 0,62±0,007 a 8571 8 0,44±0,025 a 8580 5 0,72±0,019 a 8569 10 0,45±0,004 a F-value P-level 10,560 0,000 F-value P-level 2,332 0,000 PERIOD I PERIOD II APX Enzyme Activity (EU/mg protein) Clone No. Pouches No. X±Sx Groups Clone No. Pouches No. X±Sx Groups 8566 3 0,017±0,002 f 8560 4 0,030±0,004 d 8583 6 0,027±0,011 ef 8586 7 0,043±0,002 cd 8577 6 0,032±0,009 def 8584 4 0,046±0,008 bcd 8578 6 0,032±0,006 def 8577 6 0,059±0,012 abcd 8582 4 0,032±0,007 def 8579 4 0,060±0,008 abcd 8574 4 0,036±0,01 def 8562 8 0,060±0,012 abcd 8567 4 0,041±0,01 def 8583 6 0,062±0,008 abcd 8568 4 0,044±0,007 def 8566 3 0,063±0,009 abcd 8587 5 0,60±0,018 cdef 8570 8 0,063±0,008 abcd 8585 4 0,071±0,01 bcdef 8564 5 0,071±0,008 abcd 8572 3 0,073±0,01 bcdef 8580 5 0,072±0,012 abcd PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9135 8575 4 0,081±0,03 bcdef 8563 4 0,073±0,009 abcd 8561 6 0,082±0,01 bcdef 8565 3 0,073±0,007 abcd 8565 3 0,091±0,03 bcdef 8578 6 0,074±0,018 abcd 8584 4 0,094±0,02 abcdef 8576 3 0,075±0,006 abcd 8576 3 0,096±0,03 abcdef 8574 4 0,075±0,006 abcd 8571 8 0,097±0,01 abcdef 8569 10 0,078±0,014 abcd 8569 10 0,103±0,01 abcdef 8585 4 0,078±0,016 abcd 8586 7 0,107±0,03 abcdef 8561 6 0,079±0,013 abcd 8560 4 0,112±0,03 abcde 8572 3 0,079±0,021 abcd 8570 8 0,120±0,02 abcde 8582 4 0,079±0,020 abcd 8564 5 0,124±0,02 abcd 8573 8 0,079±0,031 abcd 8579 4 0,124±0,03 abcd 8567 4 0,080±0,009 abcd 8563 4 0,139±0,02 abc 8575 4 0,082±0,018 abcd 8573 8 0,139±0,04 abc 8568 4 0,086±0,025 abc 8581 4 0,156±0,02 ab 8581 4 0,088±0,020 abc 8580 5 0,164±0,04 ab 8587 5 0,098±0,033 ab 8562 8 0,184±0,05 a 8571 8 0,104±0,013 a F-value P-level 2,821 0,000 F-value P-level 1,000 0,470 Significant effects of sampling period and clone factors on chlorophyll-a (cl-a) levels were determined (P < 0.05), whereas the effects of pouch number and pouch number × clone interaction were not statistically significant (P > 0.05). Chlorophyll-a concentration showed a seasonal decrease from February to August. For chlorophyll-b (kl-b) levels, only the sampling period factor was significant (P < 0.05), the effect of other factors was not statistically significant (P > 0.05). Chlorophyll-b concentrations similarly decreased with the transition from February to August. Total chlorophyll content was also significantly affected by clone and sampling period factors. However, the effect of the number of sacs and the sac number × clone interaction was not statistically significant (P > 0.05). Total chlorophyll content showed a decrease from February to August. When the relationships between carotenoid concentrations and independent variables (number of pouches, sampling period, clone and number of pouches × clone interaction) were analyzed, it was determined that the sampling period and clone factors had statistically significant effects on carotenoid content (P < 0.05). On the other hand, sac number and sac number × clone interaction had no statistically significant effect on carotenoid content (P >0.05) (Table 2). When the seasonal variation was analyzed, it was observed that carotenoid content decreased significantly in August compared to February. According to the analysis of variance (ANOVA) and Duncan's Multiple Comparison Test, homogeneous groups for the sampling periods (February and August) for chlorophyll-a, chlorophyll-b, total chlorophyll, and carotenoid amounts are presented in Table 4. Table 4. Variation in Photosynthetic Pigment (Chlorophyll-a, Chlorophyll-b, Total Chlorophyll, and Carotenoids) Concentrations in Different Pinus brutia Clones in I- (February) and II- (August) Periods PERIOD I PERIOD II Chlorophyll A (mg/g) Clone No. Pouches No. X±Sx Groups Clone No. Pouches No. X±Sx Groups 8583 6 0,05±0,005 e 8575 4 0,0386±0,003 I 8582 4 0,05±0,006 e 8563 4 0,0396±0,002 ıi 8564 5 0,05±0,004 e 8587 5 0,0431±0,002 hıi 8572 3 0,05±0,005 de 8564 5 0,0451±0,006 ghıi PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9136 8560 4 0,06±0,005 cde 8576 3 0,0455±0,003 ghıi 8562 8 0,06±0,005 cde 8585 4 0,0481±0,003 fghıi 8587 5 0,06±0,005 cde 8565 3 0,0488±0,003 efghı 8586 7 0,06±0,005 cde 8581 4 0,0489±0,002 efghı 8579 4 0,06±0,006 bcde 8572 3 0,0514±0,001 defgh 8570 8 0,06±0,0055 bcde 8583 6 0,0515±0,005 defgh 8584 4 0,06±0,006 bcde 8579 4 0,0526±0,00059 cdefg 8565 3 0,06±0,007 abcde 8582 4 0,053±0,002 cdefg 8561 6 0,06±0,007 abcde 8567 4 0,0532±0,005 cdefg 8563 4 0,06±0,005 abcde 8571 8 0,0549±0,003 bcdefg 8580 5 0,06±0,006 abcde 8568 4 0,0563±0,004 bcdef 8578 6 0,07±0,006 abcde 8586 7 0,0564±0,003 bcdef 8575 4 0,07±0,007 abcde 8560 4 0,0568±0,0007 bcdef 8576 3 0,07±0,004 abcde 8580 5 0,0572±0,003 bcdef 8581 4 0,07±0,006 abcde 8574 4 0,0579±0,005 bcdef 8566 3 0,07±0,003 abcde 8562 8 0,0581±0,002 bcdef 8571 8 0,07±0,008 abcde 8569 10 0,0594±0,001 bcde 8577 6 0,07±0,005 abcde 8577 6 0,06±0,0041 abcd 8568 4 0,07±0,007 abcd 8570 8 0,0603±0,003 abcd 8573 8 0,07±0,005 abcd 8566 3 0,0608±0,002 abcd 8585 4 0,08±0,006 abc 8584 4 0,615±0,0057 abcd 8574 4 0,08±0,006 abc 8578 6 0,6251±0,004 abc 8567 4 0,08±0,006 ab 8561 6 0,0652±0,0009 ab 8569 10 0,08±0,007 a 8573 8 0,0701±0,001 a F-value P-level 2,025 0,003 F-value P-level 5,831 0,000 PERIOD I PERIOD II Chlorophyll B (mg/g) Clone No. Pouches No. X±Sx Groups Clone No. Pouches No. X±Sx Groups 8564 5 0,03±0,003 e 8563 4 0,0174±0,0013 h 8583 6 0,03±0,002 e 8576 3 0,0195±0,0011 gh 8582 4 0,03±0,004 de 8564 5 0,0204±0,0032 fgh 8572 3 0,03±0,003 de 8587 5 0,0205±0,0014 fgh 8562 8 0,03±0,003 cde 8574 4 0,0217±0,0061 efgh 8570 8 0,03±0,004 cde 8567 4 0,0202±0,0035 defgh 8575 4 0,03±0,004 cde 8581 4 0,0223±0,0009 cdefgh 8584 4 0,03±0,005 cde 8583 6 0,0224±0,0023 cdefgh 8565 3 0,03±0,003 bcde 8582 4 0,0225±0,0007 bcdefgh 8579 4 0,03±0,005 bcde 8585 4 0,0227±0,0015 bcdefgh 8581 4 0,03±0,002 bcde 8572 3 0,0228±0,0014 bcdefgh 8560 4 0,03±0,004 bcde 8579 4 0,0244±0,001 abcdefgh 8563 4 0,04±0,004 abcde 8580 5 0,0247±0,0008 abcdefg 8587 5 0,04±0,003 abcde 8569 10 0,0252±0,0002 abcdefg 8576 3 0,04±0,005 abcde 8560 4 0,0256±0,0006 abcdefg 8580 5 0,04±0,003 abcde 8568 4 0,0257±0,0013 abcdefg 8561 6 0,04±0,004 abcde 8575 4 0,0263±0,0027 abcdefg 8571 8 0,04±0,004 abcde 8586 7 0,0268±0,001 abcdefg 8566 3 0,04±0,004 abcde 8571 8 0,0269±0,0006 abcdefg 8578 6 0,04±0,003 abcde 8584 4 0,0272±0,0028 abcdef 8577 6 0,04±0,004 abcde 8565 3 0,0273±0,0039 afbcdef 8573 8 0,04±0,004 abcde 8577 6 0,0275±0,0021 abcde 8585 4 0,04±0,003 abcde 8570 8 0,0288±0,00085 abcde 8574 4 0,04±0,003 abcd 8561 6 0,0292±0,0005 abcd 8568 4 0,04±0,004 abcd 8562 8 0,096±0,0017 abc 8586 7 0,05±0,004 abc 8578 6 0,0299±0,0013 ab 8569 10 0,05±0,006 ab 8566 3 0,0304±0,0015 a 8567 4 0,05±0,007 a 8573 8 0,0308±0,0014 a F-value P-level 2,777 0,000 F-value P-level 1,908 0,006 PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9137 PERIOD I PERIOD II Total Chlorophyll (mg/g) Clone No. Pouches No. X±Sx Groups Clone No. Pouches No. X±Sx Groups 8583 6 0,08±0,007 f 8563 4 0,0569±0,0034 i 8564 5 0,08±0,007 ef 8587 5 0,0636±0,0037 ıi 8582 4 0,08±0,010 def 8575 4 0,0649±0,0014 ıi 8572 3 0,09±0,008 cdef 8576 3 0,065±0,0039 ıi 8562 8 0,10±0,007 cdef 8564 5 0,0655±0,003 hıi 8560 4 0,10±0,010 cdef 8585 4 0,0709±0,0043 ghıi 8570 8 0,10±0,009 cdef 8581 4 0,0712±0,0029 fghıi 8584 4 0,10±0,010 cdef 8583 6 0,0739±0,0068 efghı 8579 4 0,10±0,011 cdef 8572 3 0,0742±0,0025 efghı 8587 5 0,10±0,008 cdef 8567 4 0,0752±0,0081 defghı 8565 3 0,10±0,010 cdef 8582 4 0,0755±0,0027 defghı 8575 4 0,10±0,010 bcdef 8565 3 0,0761±0,0066 cdefghı 8563 4 0,11±0,009 abcdef 8579 4 0,0769±0,0015 cdefghı 8561 6 0,11±0,011 abcdef 8574 4 0,0795±0,0095 bcdefghı 8580 5 0,11±0,008 abcdef 8571 8 0,0818±0,0038 bcdefgh 8581 4 0,11±0,008 abcdef 8580 5 0,0819±0,0032 bcdefgh 8576 3 0,11±0,009 abcdef 8568 4 0,082±0,0051 bcdefgh 8586 7 0,11±0,009 abcdef 8560 4 0,0824±0,0012 bcdefg 8578 6 0,11±0,009 abcdef 8586 7 0,0832±0,0039 bcdefg 8571 8 0,11±0,011 abcdef 8569 10 0,0846±0,0009 bcdefg 8566 3 0,11±0,006 abcde 8577 6 0,0875±0,0062 abcdefg 8577 6 0,12±0,008 abcd 8562 8 0,0878±0,0039 abcdef 8573 8 0,12±0,009 abc 8584 4 0,0888±0,0085 abcde 8568 4 0,12±0,011 abc 8570 8 0,0891±0,0037 abcde 8585 4 0,12±0,009 abc 8566 3 0,0912±0,0031 abcd 8574 4 0,12±0,008 abc 8578 6 0,0924±0,0053 abc 8569 10 0,14±0,012 ab 8561 6 0,0947±0,0014 ab 8567 4 0,14±0,010 a 8573 8 0,103±0,0029 a F-value P-level 2,163 0,001 F-value P-level 4,5957 0,000 PERIOD I PERIOD II Carotenoid (mg/g) Clone No. Pouches No. X±Sx Groups Clone No. Pouches No. X±Sx Groups 8582 4 5,75±0,45 h 8563 4 4,76±0,25 h 8564 5 5,84±0,35 gh 8576 3 4,9±0,27 gh 8583 6 5,96±0,29 efgh 8564 5 5,44±0,7 fgh 8560 4 6,19±0,31 defgh 8565 3 5,49±0,24 efgh 8586 7 6,31±0,41 defgh 8587 5 5,51±0,34 defgh 8572 3 6,36±0,38 defgh 8583 6 5,57±0,50 defgh 8570 8 6,39±0,42 defgh 8582 4 5,75±0,38 cdefgh 8587 5 6,40±0,39 defgh 8581 4 5,76±0,09 cdefgh 8580 5 6,44±0,46 cdefgh 8567 4 5,85±0,71 cdefgh 8562 8 6,74±0,38 bcdefgh 8575 4 5,97±0,06 cdefgh 8579 4 6,82±0,52 bcdefgh 8572 3 5,99±0,33 cdefgh 8584 4 6,87±0,56 abcdefgh 8574 4 5,99±0,86 cdefgh 8563 4 6,99±0,47 abcdefgh 8580 5 6,06±0,15 cdefg 8561 6 7,03±0,66 abcdefgh 8579 4 6,27±0,05 cdef 8576 3 7,03±0,32 abcdefgh 8585 4 6,29±0,42 cdef 8565 3 7,04±0,56 abcdefgh 8584 4 6,36±0,56 bcdef 8581 4 7,09±0,36 abcdefgh 8571 8 6,4±0,06 abcdef 8575 4 7,12±0,52 abcdefgh 8568 4 6,41±0,15 abcdef 8566 3 7,34±0,35 abcdefg 8560 4 6,63±0,15 abcdef 8577 6 7,44±0,23 abcdef 8570 8 6,67±0,18 abcdef 8571 8 7,46±0,54 abcdef 8577 6 6,71±0,46 abcdef PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9138 8568 4 7,67±0,54 abcde 8586 7 6,74±0,13 abcde 8578 6 7,72±0,42 abcde 8569 10 6,8±0,02 abcd 8567 4 7,75±0,55 acbd 8578 6 6,9±0,32 abc 8573 8 7,97±0,48 abc 8561 6 7,008±0,18 abc 8585 4 8,00±0,45 ab 8562 8 7,02±0,26 abc 8574 4 8,07±0,38 ab 8573 8 7,58±0,35 ab 8569 10 8,39±0,34 a 8566 3 7,64±0,36 a F-value P-level 2,555 0,000 F-value P-level 3,580 0,000 DISCUSSION The findings of this study showed that photosynthetic pigment concentrations (chlorophyll-a, chlorophyll-b, total chlorophyll, and carotenoids) in Pinus brutia needle leaves were significantly affected not only by abiotic environmental factors but also by biotic stress factors caused by the pine processionary moths (Thaumetopoea pityocampa and Thaumetopoea wilkinsoni). In particular, an increase in photosynthetic pigment levels was observed in February, the active feeding period of pine processionary moth larvae. Thaumetopoea spp. cause defoliation of P. brutia individuals through their feeding activities in winter and early spring (intensively in February-March). This defoliation is an important biotic stress factor that can decrease tree growth performance and mortality in young plantations in cases of severe infection (Carus 2004; Battisti et al. 2005; Kanat et al. 2005). The results of this study revealed that chlorophyll-a (chl-a) concentration in Pinus brutia needles was significantly affected by both sampling period and clone factors. In contrast, chlorophyll-b (cl-b) concentration was significantly affected only by sampling period factor. Chlorophyll levels were found to be significantly higher in the first sampling period (February), when the impact of pine processionary moth (Thaumetopoea spp.) was particularly intense, compared to the second period (August). The highest chl-a (0.08 mg/g wet weight) and total chlorophyll (0.14 mg/g wet weight) contents in clone N8569 (10 pouches), which had the highest number of pouches in the same period, support the hypothesis that biotic stress caused by pine processionary moth may induce pigment biosynthesis as a defense mechanism in plants. These findings are in agreement with the literature that plants use pigment production as an adaptation strategy to optimize their photosynthetic capacity under stress conditions. For example, Tanaka and Tanaka (2011) reported that chlorophyll-a and chlorophyll-b pigments can interconvert in response to environmental stresses. This dynamic conversion is a physiological adaptation mechanism to exogenous stress signals. Similarly, Nouri et al. (2023) emphasized that genotypes tolerant to stress conditions generally have higher chlorophyll and carotenoid contents, which increases the overall resilience of plants against biotic and abiotic stresses. In this context, the high pigment levels observed in individuals with high sac counts in the present study can be interpreted as a physiological response to biotic damage. Changes in photosynthetic pigment concentrations between February and August also reflect the significant effects of abiotic environmental factors. Sauceda et al. (2008) reported that the observed variations in chlorophyll content were closely related to abiotic stress factors such as water stress and high light intensity. Increased temperature and light intensity in summer can inhibit the biosynthesis of photosynthetic pigments, leading to a decrease in chlorophyll and carotenoid levels (Yordanov et al. 2000; Pukacki and Kamińska-Rożek 2005). In this study, a significant decrease in chlorophyll and carotenoid PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9139 levels was generally detected in August compared to February (Table 2, Table 4). Brett and Singer (1973) also stated that high light and temperature conditions may decrease chlorophyll content. However, it can be concluded that this seasonal variation in this study is largely due to environmental factors and that the damage by the pine processionary moth (Thaumetopoea spp.) has an increasing effect on pigment biosynthesis. Therefore, it is thought that significant differences emerged between the sampling periods and pigment concentrations obtained in the first period (February) were higher than in the second period (August). A similar trend was observed for carotenoid concentrations. Statistical analyses revealed that sampling period and clone factors significantly affected carotenoid levels. Carotenoid levels were significantly higher in February compared to August. Carotenoids are important antioxidant molecules in protecting chlorophyll against photooxidative damage and detoxification of reactive oxygen species (ROS), as well as functioning as auxiliary pigments in the photosynthetic antenna system (Zhang et al. 2021). These properties play a critical role in the defense mechanisms of plants against biotic stress factors such as pine processionary moth (Thaumetopoea spp.). Nouri et al. (2023) also provided evidence supporting these findings, stating that genotypes tolerant to stress conditions generally have higher levels of carotenoids. The fact that both chlorophyll and carotenoid concentrations were found to be high in February, when pine processionary moth (Thaumetopoea spp.) damage was evident, suggests that biotic stress has an up-regulating effect on pigment biosynthesis in Pinus brutia individuals. This may be considered as an important component of the defense mechanisms developed by the plant against herbivory. The observed variability in photosynthetic pigment levels as a result of synergistic or antagonistic interactions of biotic and abiotic stressors is critical for developing a deeper understanding of the complex stress physiology of plants. As a result of examining the relationships between enzymatic antioxidants (SOD, POD, CAT, and APX) and pine processionary moth pouch number, sampling period, clone and pouch number × clone interactions, a significant positive correlation was found between pouch number and sampling period on APX activity. The CAT activity was significantly affected by the number of pouches, sampling period and clone factors, while SOD and POD activities were significantly correlated only with the sampling period factor (Table 1). Literature reviews show limited studies on enzymatic antioxidant responses in Pinus brutia. Plants increase their survival probability by activating defense mechanisms against biotic stressors such as herbivorous insects. One of these defense mechanisms is the increased activity of enzymatic antioxidant systems triggered by the production of reactive oxygen species (ROS). Superoxide dismutase dismutates the superoxide radical (O2−) into hydrogen peroxide (H2O2), increasing the tolerance of plants to oxidative stress, while the POD catalyzes the oxidation of phenolic compounds using H2O2 (Katyshev et al. 2006; Boguszewska et al. 2010). These antioxidant enzymes protect against potential damage caused by oxidative damage in plant cells (Hashemi 2019). Biotic stressors such as herbivorous insects enhance defense mechanisms against oxidative stress by increasing the activities of SOD, POD, CAT, and APX in plants. These enzymatic responses play an important role in enhancing the physiological responses of plants to biotic stress and thus their survival capacity (Xu et al. 2015). In this study, a significant increase in enzymatic antioxidant activities such as SOD, POD, CAT, and APX was observed in February when pine processionary moth (Thaumetopoea spp.) damage was effective. Skwarek et al. (2017) reported differences in enzymatic antioxidant levels PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9140 between species due to Melolontha melolontha causing root damage in Pinus sylvestris and Larix decidua species. This finding in the present study suggests that Pinus brutia individuals are more susceptible to pine processionary moth-induced biotic stress in February and therefore activate their defense mechanisms more intensively. In August, a decrease in these enzymatic activities was observed with the decrease in pine processionary moth damage. The results of the analysis revealed that all enzymatic antioxidant activities (SOD, POD, CAT, APX) showed a significant decrease from February, when pine processionary moth (Thaumetopoea spp.) damage was intense, to August, when the processionary moth effect decreased (Table 1). This finding indicates that antioxidant enzymes play a more active role against oxidative damage during the period of high biotic stress and that the activity of these enzymatic defense mechanisms decreases during the period of reduced stress. Thus, this study clearly demonstrates that a specific biotic stressor such as pine processionary moth dynamically affects the enzymatic antioxidant activities of Pinus brutia individuals, triggering their defense response and that this defense response shows seasonal changes. The results from this study revealed that CAT enzyme activity was significantly correlated with pine processionary moth pouch number, sampling period and Pinus brutia clone (Table 1). The plant plasma membrane constantly interacts with the external environment, which can activate signal transduction pathways. Biotic and abiotic stress factors can modulate ion flow by causing abrupt changes in cell membrane potential (Ebel and Mithöfer 1998; Shabala 2006). Damage signals caused by herbivorous insects can lead to generating electrical signals that propagate throughout the plant (Maffei and Bossi 2006). Hydrogen peroxide can be strongly depolarized by insect feeding (Peiffer and Felton 2005). In addition to mechanical damage, plants can recognize herbivore-specific elicitor molecules. These elicitors can be found in insect oral secretions (Halitschke et al. 2001), oviposition secretions (Voirol et al. 2020), and feces (frass) (Ray et al. 2015). In a study by Liu et al. (2019), bark processionary moths did not alter POD activity on Pinus yunnanensis but increased CAT activity. CAT plays an important role in meeting the increased energy demand of the plant under stress conditions by removing H2O2 (Kerchev et al. 2016). Moreover, H2O2 induced by salicylic acid can damage the digestive system of insects and inhibit their growth and development (Peng et al. 2004; Maffei et al. 2007). These literature findings support the significant relationship of CAT enzyme with the present study’s findings for the number of pouches, sampling period, and clone. Skwarek et al. (2017) reported that insect damage increased the activities of SOD and POD enzymes. Liu et al. (2019) observed an increase in the levels of SOD, POD, and CAT enzymes as a result of Tomicus yunnanensis Kirkendall and Faccoli and Tomicus minor Hartwig damage in their study on Pinus yunnanensis Franch. The results obtained in this study showed that only the sampling period factor was statistically significant in the relationship between SOD enzyme activity and pine processionary moth pouch number, sampling period, clone and pouch number × clone interaction (Table 1). The SOD enzyme provides a protective mechanism against cellular oxidative damage by converting superoxide radical (O2−) to hydrogen peroxide (H2O2), and this process plays a critical role in the defense responses of plants against biotic and abiotic stresses (Jabs et al. 1997). Furthermore, the enzymes SOD, POD, CAT, and APX detoxify O2−and H2O2, forming a synergistic protection mechanism against these stresses (Mittler 2002; Prattipati et al. 2021). The POD enzymes are an important group of enzymes that rapidly activate plant defense responses against insect damage and can inhibit insect PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Yilmaz et al. (2025). “Biotic stress response of pine,” BioResources 20(4), 9127-9147. 9141 growth by oxidizing phenolic compounds (War et al. 2012). Liu et al. (2019) observed an increase in SOD, POD, and CAT activities after Tomicus yunnanensis and Tomicus minor damage. Skwarek et al. (2017) found that insect damage on Pinus sylvestris and Larix decidua increased SOD enzyme activities. These literature findings support that biotic stress leads to the induction of enzymatic responses that enhance plant defense (Lamb and Dixon 1997; Keeling and Bohlmann 2006). The pine processionary moth directly damages the tree and can trigger biological defense systems, leading to more subtle weakening. The insect’s feeding behaviors, particularly chemical salivary secretions, can increase the tree’s oxidative stress levels and trigger biological responses. Such indirect effects can affect tree health long-term but may not be detectable through direct observation. Therefore, a complete understanding of the pest’s effects requires considering visible damage and the tree’s biological responses. Furthermore, trees employ tolerance to herbivore attacks, which is the ability to maintain their fitness despite damaged tissue. This tolerance encompasses both visible and more subtle mechanisms (Stowe et al. 2000). As described by the cited authors, plants can exhibit “compensatory growth” after herbivore attack, regenerate new tissue, increase photosynthetic capacity, or compensate for the damage by storing nutrients. However, the real secret underlying how plants develop resistance (tolerance) to herbivore attacks occurs in complex changes in gene expression that have not yet been fully understood (Kessler and Baldwin 2002). This study evaluated the effects of pine processionary moth (Thaumetopoea pityocampa and Thaumetopoea wilkinsoni) damage and seasonal environmental factors on photosynthetic pigment concentrations and enzymatic antioxidant activities in Pinus brutia. Results showed that pine processionary moth-induced biotic stress caused seasonal variations in chlorophyll-a, chlorophyll-b, total chlorophyll, and carotenoid levels. In particular, the increase in photosynthetic pigment levels during intense insect damage suggests the activation of plant defense mechanisms. In addition, changes in SOD, POD, CAT, and APX enzyme activities reflect the physiological responses of plants to biotic stress. The increase in the activities of these enzymes in February indicates that plant defense is strengthened during this period when biotic stress is more pronounced. CONCLUSIONS 1. Damage by the pine processionary moth (Thaumetopoea spp.) induces oxidative stress and activation of enzymatic defense systems in Pinus brutia. These findings highlight the important ecological and economic impacts of biotic damage on forestry and reveal the critical role of understanding the physiological responses of plants in controlling such pests. 2. The study found that photosynthetic pigment concentrations (chlorophylls and carotenoids) were significantly affected by pine processionary moth damage. During the moth’s intense feeding period in February, the levels of chlorophyll-a, total chlorophyll, and carotenoids were higher. This suggests that the plants activate a defense mechanism by increasing pigment production to cope with the stress. 3. Overall, the findings demonstrate a clear link between the biotic stress from the pine processionary moth and the seasonal variations in both photosynthetic pigments and antioxidant enzyme activities within the trees. 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