Abstract
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.
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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
Graphical Abstract
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 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 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 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.
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.
Table 1. Evaluation of Enzymatic Antioxidant Activities by Linear Regression Analysis
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
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.
Table 3. Variation of Enzymatic Antioxidant (SOD, POD, CAT, APX) Activities in Different Pinus brutia Clones in I- (February) and II- (August) Periods
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
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 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 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 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
- 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.
- 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.
- 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.
ACKNOWLEDGMENTS
Ergin Yilmaz, a PhD student in the Sustainable Forestry Program was supported by the Higher Education Council’s 100/2000 Scholarship at Kastamonu University, Institute of Science. This research was financially supported by the Kastamonu University Scientific Research Fund under the project number KÜ-BAP01/2020-43. As the authors, we would like to express our gratitude to Assoc. Prof. Dr. Ferhat Kara, from the Department of Silviculture, Faculty of Forestry, Kastamonu University, for his invaluable support in statistical analysis.
REFERENCES CITED
Agarwal, S., and Pandey, V. (2004). “Antioxidant enzyme responses to NaCl stress in Cassia angustifolia,” Biologia Plantarum 48(4), 555-560. DOI: 10.1023/B:BIOP.0000047152.07878.e7
Anetsos, P. K. (1981). “Monograph of Pinus halepensis (Mill) and Pinus brutia Ten,” Annales Forestales 9(2), 39-77.
Anonim (Dpt) (1995). Forestry Report Devlet Planlama Teşkilatı, Ankara. P.183.
Arbez, M. (1974). “Distribution, ecology and variation of Pinus brutia in Turkey,” Forest Genetic Resources Information 3, FAO Rome: 21-2
Arnon, D. I. (1949). “Copper enzymes in isolated chloroplast Polyphenoloxidase in Beta vulgaris,” Plant Physiology 24, 1-10. DOI: 10.1104/pp.24.1.1
Avci, M. (2000). “Investigation on structure of egg-batches, parasitism and egg laying habits of Thaumetopoea pityocampa (Den.&Schiff.) (Lepidoptera: Thaumetopoeidae) in various regions of Turkey,” Turkey Journals of Entomology 24, 167-178.
Battisti, A., Stastny, M., Netherer, S., Robinet, C., Schopf, A., Roques, A., and Larsson, S. (2005). “Expansion of geographic range in the pine processionary moth caused by increased winter temperatures,” Ecological Applications 15(6), 2084-2096. DOI: 10.1890/04-1903
Boguszewska, D., Grudkowska, M., and Zagdańska, B. (2010). “Drought-responsive antioxidant enzymes in potato (Solanum tuberosum L.),” Potato Research 53, 373-382. DOI: 10.1007/s11540-010-9178-6
Brett, W. J., and Singer, A. C. (1973). “Chlorophyll concentration in leaves of Juniperus virginiana L., measured over a 2-year period,” American Midland Naturalist 194-200. DOI: 10.2307/2424280
Carus, S. (2004). “Impact of defoliation by the pine processionary moth (Thaumetopoea pityocampa) on radial, height and volume growth of Calabrian pine (Pinus brutia) trees in Turkey,” Phytoparasitica 32, 459-469. DOI: 10.1007/BF02980440
Critchfield, W. B., and Little, E. L. (1966). “Geographic distribution of the pines of the world (No. 991),” US Department of Agriculture, Forest Service. DOI: 10.5962/bhl.title.66393
Çanakçıoğlu, H., (1993). “Orman Entomolojisi (Özel Bölüm), İstanbul Üniversitesi Yayın No: 3623,” İstanbul Üniversitesi Fakülte Yayınları No: 382, 385s.
Denis, J. M., and Schiffermüller, I. (1776). Systematisches Verzeichniß der Schmetterlinge der Wienergegend herausgegeben von einigen Lehrern am K. K. Theresianum, Wien, S.1–323.Taf.Ia, Ib.
Devkota, B., and Schmidt, G. H. (1990). “Larval development of Thaumetopoea pityocampa (Den. & Schiff.) (Lep., Thaumetopoeidae) from Greece as influenced by different host plants under laboratory conditions 1,” Journal of Applied Entomology 109(1‐5), 321-330. DOI: 10.1111/j.1439-0418.1990.tb00059.x
DPT. (2001). “Sekizinci Beş Yıllık Kalkınma Planı, Ormancılık Özel İhtisas Komisyonu Raporu, Devlet Planlama Teşkilatı, yayın no:2531,” Özel İhtisas Komisyonu 547, Ankara.
Durkaya, A., Durkaya, B., and Dal, İ. (2009). “Length Research Paper The effects of the pine processionary moth on the increment of Crimean pine trees in Bartin, Turkey,” African Journal of Biotechnology 8(10).
Ebel, J., and Mithöfer, A. (1998). “Early events in the elicitation of plant defense,” Planta 206, 335-348. DOI: 10.1007/s004250050409
Fady, B., Lefèvre, F., Reynaud, M., Vendramin, G.G., Dagher-Kharrat, M.B., Anzidei, M., and Bariteau, M. (2003). “Gene flow among different taxonomic units: evidence from nuclear and cytoplasmic markers in Cedrus plantation forests,” Theoretical and Applied Genetics 107(6), 1132-1138. DOI: 10.1007/s00122-003-1323-z
Gong, Y., Toivonen, P. M., Lau, O. L., and Wiersma, P. A. (2001). “Antioxidant system level in ‘Braeburn’ apple is related to its browning disorder,” Botanical Bulletin of Academia Sinica 42, 259-264.
Gulsen, O., Eickhoff, T., Heng-Moss, T., Shearman, R., Baxendale, F., Sarath, G., and Lee, D. (2010). 42, 259-264. “Characterization of peroxidase changes in resistant and susceptible warm-season turfgrasses challenged by Blissus occiduus,” Arthropod-Plant Interactions 4, 45-55. DOI: 10.1007/s11829-010-9086-3
Halitschke, R., Schittko, U., Pohnert, G., Boland, W., and Baldwin, I. T. (2001). “Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. III. Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore-specific plant responses,” Plant Physiology 125(2), 711-717. DOI: 10.1104/pp.125.2.711
Hare, J. D. (2011). “Ecological role of volatiles produced by plants in response to damage by herbivorous insects,” Annual Review of Entomology 56(1), 161-180. DOI: 10.1146/annurev-ento-120709-144753
Hashemi, S. (2019). “Effect of nanoparticles on lipid peroxidation in plants,” in: Advances in Lipid Metabolism, IntechOpen, London, UK. DOI: 10.5772/intechopen.88202
Howe, G. A., and Jander, G. (2008). “Plant immunity to insect herbivores,” Annual Review of Plant Biology 59(1), 41-66. DOI: 10.1146/annurev.arplant.59.032607.092825
Jabs, T., Tschöpe, M., Colling, C., Hahlbrock, K., and Scheel, D. (1997). “Elicitor-stimulated ion fluxes and O2− from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley,” Proceedings of the National Academy of Sciences 94(9), 4800-4805. DOI: 10.1073/pnas.94.9.4800
Kanat, M., and Türk, M. (2002). “New cage method for struggling against Thaumetopoea pityocampa (Schiff), 109-114,” in: Proceedings of Pine Processionary Moth Symposium, M. Kanat (Ed), 24-25 April 2002, Kahramanmaraş, Turkey, 226 pp.
Kanat, M., Alma, M. H., and Sivrikaya, F. (2005). “Effect of defoliation by Thaumetopoea pityocampa (Den. & Schiff.) (Lepidoptera: Thaumetopoeidae) on annual diameter increment of Pinus brutia Ten. in Turkey,” Annals of Forest Science 62(1), 91-94. DOI: 10.1051/forest:2004095
Kara, N., Korol, L., Isik, K. and Schiller G. (1997). “Genetic diversity in Pinus brutia Ten.: Altitudinal variation,” Silvae Genetica 46 (2-3), 155-1 61.
Katyshev, A. I., Konstantinov, Y. M., and Kobzev, V. F. (2006). “Characterization of Mn-and Cu/Zn-containing superoxide dismutase gene transcripts in Larix gmelinii,” Molecular Biology 40, 327-329. DOI: 10.1134/S0026893306020208
Kaya, Z., and Raynal, D. J. (2001). “Biodiversity and conservation of Turkish forests,” Biological Conservation 97(2), 131-141. DOI: 10.1016/S0006-3207(00)00069-0
Keeling, C. I., and Bohlmann, J. (2006). “Genes, enzymes and chemicals of terpenoid diversity in the constitutive and induced defense of conifers against insects and pathogens,” New Phytologist 170(4), 657-675. DOI: 0.1111/j.1469-8137.2006.01716.x
Kerchev, P., Waszczak, C., Lewandowska, A., Willems, P., Shapiguzov, A., Li, Z., and Van Breusegem, F. (2016). “Lack of glycolate oxıdase1, but not glycolate oxıdase2, attenuates the photorespiratory phenotype of catalase2-deficient Arabidopsis,” Plant Physiology 171(3), 1704-1719. DOI: 10.1104/pp.16.00359
Kessler, A., and Baldwin, I. T. (2002). “Plant responses to insect herbivory: the emerging molecular analysis,” Annual Review of Plant Biology 53(1), 299-328. DOI: 10.1146/annurev.arplant.53.100301.135207
Lamb, C., and Dixon, R. A. (1997). “The oxidative burst in plant disease resistance,” Annual Review of Plant Biology 48(1), 251-275. DOI: 10.1146/annurev.arplant.48.1.251
Leblebici, S. E. M. A., Bozca, F. D., Topkara, E. F., and Yanar, O. (2023). “Comparison of physiological responses in some Pinus species attacked by pine processionary moth,” Russian Journal of Plant Physiology 70(6), 143. DOI: 10.1134/S1021443723601465
Liu, J., Chen, H., Wang, J., Chen, X., Yang, Z., and Liang, J. (2019). “Photosynthetic traits and antioxidative defense responses of Pinus yunnanensis after joint attack by bark beetles Tomicus yunnanensis and T. minor,” Journal of Forestry Research 30(6), 2031-2038. DOI: 10.1007/s11676-018-0844-x
Maffei, M. E., Mithöfer, A., and Boland, W. (2007). “Insects feeding on plants: rapid signals and responses preceding the induction of phytochemical release,” Phytochemistry 68(22-24), 2946-2959. DOI: 10.1016/j.phytochem.2007.07.016
Maffei, M., and Bossi, S. (2006). “Electrophysiology and plant responses to biotic stress,” in: Plant Electrophysiology: Theory and Methods, Springer Berlin Heidelberg, pp. 461-481. DOI: 10.1007/978-3-540-37843-3_20
Masutti, L., and Battisti, A. (1990). “Thaumetopoea pityocampa (Den. & Schiff.) in Italy Bionomics and perspectives of integrated control 1 2,” Journal of Applied Entomology 110(1‐5), 229-234. DOI: 10.1111/j.1439-0418.1990.tb00117.x
Michelozzi, M., Tognetti, R., Maggino, F., and Radicati, M. (2008). “Seasonal variations in monoterpene profiles and ecophysiological traits in Mediterranean pine species of group “halepensis”,” iForest-Biogeosciences and Forestry 1(1), article 65. DOI: 10.3832/ifor0206-0010065
Milios, E., Kitikidou, K., and Radoglou, K. (2019). “New silvicultural treatments for conifer peri-urban forests having broadleaves in the understory-the first application in the peri-urban of Xanthi in northeastern Greece,” South-east European forestry Seefor 10(2), 107-116. DOI: 10.15177/seefor.19-16
Mittler, R. (2002). “Oxidative stress, antioxidants and stress tolerance,” Trends in Plant Science 7(9), 405-410. DOI: 1016/S1360-1385(02)02312-9
Mushtaq, Z., Faizan, S., and Gulzar, B. (2020). “Salt stress, its impacts on plants and the strategies plants are employing against it: A review,” Journal of Applied Biology and Biotechnology 8(3), 81-91. DOI: 10.7324/JABB.2020.80315
Myteberi, I., Lushaj, B., Laçeja, F., Metaliaj, R., Malsia, V., Mine, V., and Petto, A. (2013). “The presence and the activity of egg-parasitoids of pine processionary moth (Thaumetopoea pityocampa Den & Schiff) in new forests of Austrian pine (Pinus nigra Arn.) in Korça, Kolonja e Pogradec districts, Albania,” Online International Interdisciplinary Research Journal 3(6), 34-42.
Nahal, I. (1983). “Le Pin brutia (Pinus brutia Ten. subsp. brutia). 1ère partie,” Foret Mediterraneenne (2), 165-172.
Nakano, Y., and Asada, K. (1981). “Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts,” Plant and Cell Physiology 22(5), 867-880. DOI: 10.1093/oxfordjournals.pcp.a076232
Nouri, K., Nikbakht, A., Haghighi, M., Etemadi, N., Rahimmalek, M., and Szumny, A. (2023). “Screening some pine species from North America and dried zones of western Asia for drought stress tolerance in terms of nutrients status, biochemical and physiological characteristics,” Frontiers in Plant Science 14, article 1281688. DOI: 10.3389/fpls.2023.1281688
Oppenheimer, H. R. (1967). “Mechanisms of Drought Resistance in Conifers of the Mediterranean Zone and the Arid West of the USA,” Part 1: Physiological and Anatomical Investigations, Final Report on project No. A10−FS 7, Grant No FG−Is−119, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot, Israel.
Orozco-Cardenas, M., and Ryan, C. A. (1999). “Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway,” Proceedings of the National Academy of Sciences 96(11), 6553-6557. DOI: 10.1073/pnas.96.11.6553
Paniagua Voirol, L. R., Valsamakis, G., Lortzing, V., Weinhold, A., Johnston, P. R., Fatouros, N. E., and Hilker, M. (2020). “Plant responses to insect eggs are not induced by egg‐associated microbes, but by a secretion attached to the eggs,” Plant, Cell & Environment 43(8), 1815-1826. DOI: 10.1111/pce.13746
Peiffer, M., and Felton, G. W. (2005). “The host plant as a factor in the synthesis and secretion of salivary glucose oxidase in larval Helicoverpa zea. Archives of ınsect biochemistry and physiology,” Published in collaboration with the Entomological Society of America 58(2), 106-113. DOI: 10.1002/arch.20034
Peng, J., Deng, X., Jia, S., Huang, J., Miao, X., and Huang, Y. (2004). “Role of salicylic acid in tomato defense against cotton bollworm, Helicoverpa armigera, Hubner,” Zeitschrift für Naturforschung C 59(11-12), 856-862. DOI: 10.1515/znc-2004-11-1215
Powell, G., Tosh, C. R., and Hardie, J. (2006). “Host plant selection by aphids: behavioral, evolutionary, and applied perspectives,” Annual Review of Entomology 51(1), 309-330. DOI: 10.1146/annurev.ento.51.110104.151107
Prattipati, S. D., Botcha, S., and Maradana, T. N. (2021). “Activities of antioxidant systems during germination of Sterculia urens Roxb. Seeds,” Vegetos 34, 882-888. DOI: 10.1007/s42535-021-00252-8
Pukacki, P. M., and Kamińska-Rożek, E. (2005). “Effect of drought stress on chlorophyll a fluorescence and electrical admittance of shoots in Norway spruce seedlings,” Trees 19, 539-544. DOI: 10.1007/s00468-005-0412-9
Quezel, P. (1979). “La région méditerranéenne française et ses essences forestières, signification écologique dans le contexte circum-Mediterranean,” Forêt Méditerranéenne 1(1), 7-18.
Quézel, P. (1985). “Les pins du groupe Halepensis: Ecologie vegetation ecophysiologie,” in: Le pin d’alep et le Pin brutia dans la Sylviculture Méditerranéenne, Paris, France: Options Méditerranéennes Série Etudes 86, 11-66.
Quézel, P. (2000). “Taxonomy and biogeography of mediterranean pines (Pinus halepensis and P. brutia),” in: Ecology, Biogeography and Management of Pinus halepensis and P. brutia Forest Ecosystems in the Mediterranean Basin, G. Ne’Eman and L. Trabaud (eds.), Buckhuys Publishers, Leiden 1-12. DOI: 10.2307/3236684
Ray, S., Gaffor, I., Acevedo, F. E., Helms, A., Chuang, W. P., Tooker, J., Felton, G. W., and Luthe, D. S. (2015). “Maize plants recognize herbivore-associated cues from caterpillar frass,” Journal of Chemical Ecology 41, 781-792. DOI: 10.1007/s10886-015-0619-1
Rodríguez-Mahillo, A. I., González-Muñoz, M., Vega, J. M., López, J. A., Yart, A., Kerdelhué, C., Camafeita, E., García Ortiz, J. C., Vogel, H., and Toffolo, E. P. (2012). “Setae from the pine processionary moth (Thaumetopoea pityocampa) contain several relevant allergens,” Contact Dermatitis 67(6), 367-374. DOI: 10.1111/j.1600-0536.2012.02107.x
Sauceda, J. U., Rodriguez, H. G., Lozano, R. R., Silva, I. C., Meza, M. G., and Larga, L. (2008). “Seasonal trends of chlorophylls a and b carotenoids in native trees and shrubs of Northeastern Mexico,” Journal of Biological Sciences 8(2), 258-267. DOI: 10.3923/jbs.2008.258.267
Selik, M. (1958). “Botanical investigation on Pinus brutia especially in comparison with Pinus halepensis,” Istanbul University Faculty of Forestry Journal 8a: 161-198.
Skwarek, M., Patykowski, J., and Witczak, A. (2017). “Changes in antioxidant enzyme activities in Pinus sylvestris and Larix decidua seedlings after Melolontha melolontha attack,” Forest Research Papers 78, 159-164. DOI: 10.1515/frp-2017-0017
Stowe, K. A., Marquis, R. J., Hochwender, C. G., and Simms, E. L. (2000). “The evolutionary ecology of tolerance to consumer damage,” Annual Review of Ecology And Systematics 31(1), 565-595. DOI: 10.1146/annurev.ecolsys.31.1.565
Tanaka, R., and Tanaka, A. (2011). “Chlorophyll cycle regulates the construction and destruction of the light-harvesting complexes,” Biochimica et Biophysica Acta (BBA)-Bioenergetics 1807(8), 968-976. DOI: 10.1016/j.bbabio.2011.01.002
Torres, M. A. (2010). “ROS in biotic interactions,” Physiologia plantarum 138(4), 414-429. DOI: 10.1111/j.1399-3054.2009.01326.x
Usha Rani, P., and Jyothsna, Y. (2010). “Biochemical and enzymatic changes in rice plants as a mechanism of defense,” Acta Physiologiae Plantarum 32, 695-701. DOI: 10.1007/s11738-009-0449-2
Usta, H. F. (1990). “Kızılçam (Pinus brutia Ten.) Ağaçlandırmalarında hasılat Araştırmaları. A study on the yield of Pinus brutia Ten. Plantations,” Ormancılık Araştırma Enstitüsü Yayınları Teknik Bülten 219, 138 P.
Vega, J. M., Moneo, I., Armentia, A., López-Rico, R., Curiel, G., Bartolome, B., and Fernandez, A. (1997). “Anaphylaxis to a pine caterpillar,” Allergy 52(12). DOI: 10.1111/j.1398-9995.1997.tb02532.x
War, A. R., Paulraj, M. G., Ahmad, T., Buhroo, A. A., Hussain, B., Ignacimuthu, S., and Sharma, H. C. (2012). “Mechanisms of plant defense against insect herbivores,” Plant Signaling and Behavior 7(10), 1306-1320. DOI: 10.4161/psb.21663
Witham, F. H., Blaydes, D. F., and Devlin, R. M., (1971). “Experiments in plant physiology,” Van Nostrand Reinhold Company New York, pp. 55–56.
Xu, Z., Jiang, Y., and Zhou, G. (2015). “Response and adaptation of photosynthesis, respiration, and antioxidant systems to elevated CO2 with environmental stress in plants,” Frontiers in Plant Science 6, article 701. DOI: 10.3389/fpls.2015.00701
Yee, Y., Tam, N. F. Y., Wong, Y. S., and Lu, C. Y., (2002). “Growth and physiological responses of two mangrove species (Bruguira gymnorrhiza and Kandelia candel) to waterlogging,” Environmental and Experimental Botany 1-13.
Yordanov, I., Velikova, V., and Tsonev, T. (2000). “Plant responses to drought, acclimation, and stress tolerance,” Photosynthetica 38, 171-186. DOI: 10.1023/A:1007201411474
Zhang, R. R., Wang, Y. H., Li, T., Tan, G. F., Tao, J. P., Su, X. J., and Xiong, A. S. (2021). “Effects of simulated drought stress on carotenoid contents and expression of related genes in carrot taproots,” Protoplasma 258, 379-390. DOI: 10.1007/s00709-020-01570-5
Article submitted: June 11, 2025; Peer review completed: August 9, 2025; Revised version received and accepted: August 15, 2025; Published: August 27, 2025.
DOI: 10.15376/biores.20.4.9127-9147