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
