Specimens of Ganoderma camelum were collected from Khanaspur Halipad, Abbottabad District, Khyber Pakhtunkhwa Province, Pakistan, between June 2018 and August 2019 and deposited under deposition numbers SCUF517 and SCUF518.

The morphological characterization of the fungus, including its color, shape, and other detailed features, were conducted according to the guidelines established by Corner (1983).

The thickness of the pileus was measured at the point where the width and length of the fruiting body intersect. The color of the pileus was determined using the color chart of Munsell (1975).

Microscopic analysis was executed by observing cross sections of the dried basidiomata, which were first soaked in a 5% potassium hydroxide (KOH) solution, then stained 1% Congo red, and subsequently visualized under a MX4300H compound light microscope (Meiji Techo Co., Ltd., Japan). At least 30 measurements were meticulously recorded at a magnification of 100X. For the spore measurements, 30 counts from two samples were presented as length × width (Nagy et al., 2010), with the apiculus excluded when not compressed. The microscopic characteristics were described in alignment with the methodology outlined by Cabarroi-Hernández et al. (2019).

Genomic DNA was isolated from the specimens using a modified CTAB technique, the ITS regions were analyzed and amplified using ITS1 and ITS2 primers (White et al., 1990). The PCR amplification process was performed within a 25 μL reaction volume, utilizing a master mix [DreamTaqGreen PCR Master Mix (2X), Fermentas].

The reaction mixture included 9.5 μL distilled water, 1 μL template DNA, 12.5 μL 2X PCR master mix, and 1 μL of each primer. The amplification protocol comprised 35 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 1 min, concluding with a final extension of 10 min at 72°C. The PCR products were purified and sequenced by TSINGKE Co., Ltd. (China).

The dataset included DNA sequences of the novel species, along with additional ITS sequences obtained from GenBankwww.ncbi.nlm.nih.gov/genbank/ and relevant literature. The sequences were automatically aligned using MAFFT and manually adjusted using CLUSTALW in BioEdit software. A phylogenetic tree was constructed using MEGA ver. 10 and RAxML (Hall, 1999; Katoh et al., 2019). The reliability of the tree was assessed through bootstrapping with 1,000 replicates.

Ganoderma camelum was cultured by inoculating 1 cm sterile tissue segments onto Basidiomycete-selective malt extract agar (BSMEA). The BSMEA medium was prepared following the manufacturer’s guidelines (MEA) (Difco Laboratories, Franklin Lakes, NJ), augmented with streptomycin (100 mg/L) and benomyl 95% (4 mg/L) (Loyd et al., 2018b).

Terricolous fungi were isolated using the dilution plate technique with MEA, which was supplemented with Rose Bengal (1/15,000) and chloramphenicol (50 ppm) to inhibit bacterial growth (Smith and Dawson, 1944). Post-inoculation, the plates were incubated at 27°C for 10 days. Subsequently, the emerging colonies of Trichoderma were identified and enumerated. The samples of Ganoderma under study were subsequently preserved in the Suez Canal University Fungarium (SCUF) under the deposit numbers SCUF517 and SCUF518, respectively (https://ccinfo.wdcm.org/details?regnum=1180, accessed on 12 August 2024).

The production of laccase was assessed through the placement of 5 mm-diameter disks from 7-day-old colonies onto guaiacol-supplemented agar plates (Abdel-Azeem and Salem, 2012) and by direct inoculation into modified Czapek’s agar plates. These plates were then incubated in the dark at 28°C for 7 days. The development of a pronounced brown coloration beneath and around the fungal colony was interpreted as a positive response, indicative of guaiacol oxidation (Kalra et al., 2013).

Actively growing mycelia (five pieces of 5 mm diameter) were cultivated in 100 mL of basal nutritional medium (Umar et al., 2023) in an Erlenmeyer flask (250 mL) at 28°C and 150 rpm for 8 days from freshly prepared pure cultures of Ganoderma and Trichoderma (approximately 7 days incubation at 30°C). After incubation, the fungal broth culture containing mycelia was centrifuged at 10,000 rpm at 4°C for 20 min and then filtered using Whatman filter papers. The resulting extracellular fluid supernatant contained crude laccase, which was used for further research.

The supernatant, containing crude laccase, was employed to assess enzymatic activity by measuring the oxidation of the guaiacol substrate (Gao et al., 2011). For this quantification, a 50 mM sodium acetate buffer, adjusted to a pH of 4.5, was combined with 2 mM guaiacol. A solution comprising 1.5 mL of the crude enzyme supernatant, 1 mL of the sodium acetate buffer, and 1 mL of guaiacol was vigorously mixed for 30 s and then incubated at 30°C for 10 min (Chefetz et al., 1998). After incubation, absorbance was measured at 465 nm (465 = 12,100 M⁻¹ cm⁻¹). EA = (A* V)/(t * €* v), where E.A = enzyme activity (U/mL), A = absorbance at 465 nm, V = total volume of the reaction mixture (mL), v = enzyme volume (mL), t = incubation time (min), and € = extinction coefficient ( M⁻¹ cm⁻¹) (Gao et al., 2011).

The method described by Chefetz et al. (1998) was employed to purify the laccase. The filtrate was centrifuged at 13,000 rpm for 20 min at 10°C, after which the supernatant was precipitated with ammonium sulfate. The resulting precipitates were then dialyzed and loaded onto a DEAE-Cellulose anion-exchange column, which had been equilibrated with a 10 mM sodium acetate buffer (pH 5.5).

Subsequently, the laccase fraction was collected, concentrated, and dialyzed overnight, after which 3 mL of the DEAE-purified sample was applied to the column. Post-dialysis, the purity and molecular weights of the laccase were evaluated through SDS-PAGE analysis (Durán et al., 2002) and visualized using Coomassie Brilliant Blue R-250 staining. The relative molecular mass was estimated by comparison with standard molecular weight markers.

The Ganoderma wood degradation was evaluated using rubber wood blocks, each measuring 80 mm × 50 mm × 20 mm and weighing precisely (100 g). Initially, these blocks were immersed in distilled water overnight within plastic bags and subsequently subjected to autoclaving for 45 min at a temperature of 121°C. Subsequently, 100 mL of MEA broth was administered to each block, which was then autoclaved again under identical conditions. Following a 2 min cooling period within a laminar flow hood to facilitate the adequate absorption of the medium, Ganoderma sp. cultures derived from Petri plates were finely minced and introduced into sterile plastic bags. An additional 100 mL of MEA was added to the blocks, which were then incubated at ambient temperature for 120 days (Loyd et al., 2018b).

Control blocks without Ganoderma were included. The weight of both control wood blocks and the Ganoderma-infected wood blocks was meticulously recorded every 20 days to ascertain the degree of decay inflicted by the Ganoderma species.

This experiment investigated the antagonistic relationship between Ganoderma and Trichoderma species. A 5 mm mycelium disk, each sourced from the periphery of an actively proliferating mycelium culture of both species, was excised and transferred to a distinct agar Petri plate. This disk was then permitted to propagate for 3 days at a controlled temperature of 25°C.

Control plates contained only Ganoderma cultures, and the study was replicated three times. The percentage inhibition of radial growth (PIRG) zones for both species was computed daily over 10 days, utilizing the formula delineated by Yazid et al. (2023).

In this formula, PIRG indicated “percentage inhibition of radial growth,” R₁ indicated radial growth of the Ganoderma colony in the absence of Trichoderma, whereas R₂ showed the radial growth of the Ganoderma colony in the presence of Trichoderma.

A sterile, clean glass slide was inserted into 9-cm-diameter plates to facilitate the Ganoderma-Trichoderma interaction. An autoclaved, molten MEA layer was then applied onto the slide. The 5 mm disks, excised from 1-week-old colonies on the periphery of Ganoderma and Trichoderma isolates, were positioned 3 cm apart on the MEA surface, opposite each other across the slide. To mitigate drying, a small quantity of double-distilled water was added to the plate. Subsequently, the plate was incubated at 25°C for a week. Upon the completion of the incubation, the area where Ganoderma-Trichoderma hyphae interfaced was stained with lactophenol in cotton blue, and the slide was examined under a light microscope to assess any signs of mycelial penetration and cell wall degradation that occurred during the incubation period.

The mean values and standard deviations (±SD) from three biological replicates (n = 3) were presented in the data. These triplicate data sets transformed and were subsequently subjected to an ANOVA analysis using SPSS software. The mean differences were evaluated through the HSD (Tukey’s standardized range) test, with statistical significance set at Pd ≤ 0.05.

Principal component analysis (PCA) was employed to elucidate the relationships among the investigated cases and parameters. The statistical analyses were conducted using Statistica software (version 12.0, StatSoft Inc., Tulsa, OK, United States). Principal components analysis (PCA), ANOVA, and correlation determination were all performed at a significance level of a = 0.05. The data matrix utilized for the PCA statistical analysis of the chromatographic test results comprised three columns and 11 rows. The input matrix was automatically scaled.

ITS markers were employed to ascertain the Trichoderma species responsible for the highest laccase production. The potential species were identified by constructing a phylogenetic tree, which was generated using maximum likelihood analysis. The purified fungal mycelium was found to cluster into a distinct clade, closely related to other species, with high bootstrap values confirming the robustness of the tree’s topology (Table 1). Phylogenetic analysis facilitated the clear identification of the filamentous Trichoderma species (Figure 1). Multiple sequence alignments were executed with CLUSTALW in BioEdit software, followed by manual adjustment. Subsequently, phylogenetic trees were constructed from these alignments using MEGA Version 10.0 and RAxML, with the statistical significance of the tree being evaluated through bootstrapping with 1,000 replicates. The maximum likelihood tree topology for Ganoderma is depicted, exhibiting a 98% statistical bootstrap value, which supports the identification of a novel species (Figure 2; Table 2).

This study comprised 10 Trichoderma species and one new Ganoderma species evaluated for laccase activity. The colony characteristics of each species were determined (Table 3).

Ganoderma camelum A. Umar, sp. nov. (Figures 3A–D, 4A–E).

MycoBank# 854703.

Diagnosis: In the phylogenetic tree (Figure 2), Ganoderma martinicense and G. multipileum are very closely matrixed to our new species of G. camelum. Morphologically, G. camelum is characterized by its sessile, delicate, and very soft velvety appearance of basidiomata; similarly, a sessile basidiome is found in G. martinicense, the closest species in the phylogenetic tree. The other closest species was G. multipileum, which rarely exhibits this characteristic and has stipitate basidiomata. Growth zones are found in both G. camelum and G. martinicense. The contextum is dark cinnamon brown in G. martinicense and light-brown to brown in G. multipileum, while G. camelum exhibited a soft light brown to camel brown contextum. The spores are larger in G. multipileum (7.3–) 8.0–11.5 (−12.2) × (5.3–) 5.5–7.8 (−8.3) μm and G. martinicense (8–) 8.8–10.5 (−11.3) × (5–) 5.5–7 (−7.2) μm, while smaller in G. camelum (4.7–5.2 × 2.3–3.6 μm).

Etymology: The species epithet “camelum” refers to camel brown color.

Holotype: PAKISTAN, Khyber Pakhtunkhwa, Abbottabad District, Khanspur Halipad (34^° 1′ 16 N, 73^° 25′ 40E, on the living stem of Pinus wallichiana, elevation 2,250 m above sea level, Aisha Umar, 6th June 2018, KPKHP31) (SCUF517: GenBank PP062819).

Description: Basidiomata sessile, convex, velvety, very soft, shallow waved five concentric zones, inner four zones tawny olive (10YR) to camel brown (5YR), outer zone pinkish buff (9/4R) to beige (9/6R); Margin 0.1–0.2 mm, very thin, white; Pileus 5.5–6.5 × 6.5–7.3 cm, glabrous, bumpy, verrucose, reniform, thick, margins thin, obtuse, slight radiating lines, soft flesh; Pores 110–153 × 125–130 μm, hard porous layer, yellow-brown (5YR) to tawny (10YR), subcircular or longitudinal; Tubes 0.2–0.3 mm long, min, non-stratified, ochraceous buff (6/8YR); Context 0.4–0.5 mm thick, light brown (6/6YR) soft velvety layer, beneath brown tawny (10YR) hard layer, milky cream, dry, fibrous and corky; Crustohymeniderm palissade club-shaped, clavate, pale yellow to yellow (5Y), 32.5–52.7 × 10.2–12.4 μm, smooth, double-walled, few two to three septate or few multi-septate cells; BASIDIOLES 6.5–13 × 4.5–7 μm, inverted pear-shaped to broadly clavate with big oil droplets; Basidiospores 4.7–5.2 × 2.3–3.6 μm A_L = 4.6 μm, A_W = 2.8 μm, Q = 1.63 (n = 30/1), ellipsoid with tapering ends, smooth, bitunicate, inter-walled pillars absent, laterally pointed; Hyphal System Trimitic (1) generative hyphae (septate, clamped, colorless, thin-walled, 2–3.4 μm), (2) skeletal hyphae (thick-walled, colorless, unbranched or few branches with distal end, 3.1–4.8 μm), and (3) binding hyphae (arboriform, colorless thick-walled, much-branched, 1.2–3.4 μm).

Additional specimen examined: PAKISTAN, Khyber Pakhtunkhwa, Abbottabad District, Khanspur Halipad (34^° 1′ 16 N, 73^° 25′ 40E), on the living stem of Pinus wallichiana, elevation 2,215 m above sea level, Aisha Umar, 25 August 2019, KPKHP32 (SCUF518:GenBank PP062820).

The consensus sequence of the ITS regions was subjected to a BLAST search utilizing the NCBI GenBank database, facilitating a comparison with a sequence database to ascertain species-level taxonomic data. An unknown or novel species is identified by situating it within an evolutionary context alongside other homologous sequences through phylogenetic analyses. The initial BLAST results for our sequences and consensus yielded 100 NCBI BLAST hits, of which over 58 were designated merely as “Ganoderma sp.” (merely the genus name was provided without specifying the species). These 58 sequences of Ganoderma species remained unnamed before our initial BLAST analysis. Consequently, this study has assigned species names to all previously unknown sequences associated with the genus Ganoderma. In the NCBI Query Cover, our novel species matched 100% with previously unidentified Ganoderma species. The initial BLAST revealed a 99.66% identification percentage, with an accuracy length of 619 (Accession PP062820.1) and 621 (Accession PP062819.1). The maximum score and total score for our new sequences were 1,081, suggesting that this represents a species not previously described.

In the phylogenetic tree, several neighboring species are positioned in close proximity to our newly identified species. These include Ganoderma multipileum (Wang et al., 2009; Welti and Courtecuisse, 2010; Nguyen et al., 2023), G. martinicense (Loyd et al., 2018c), G. mizoramense (Crous et al., 2017), and G. parvulum (Torres-Torres and Guzmán-Dávalos, 2012) (Figure 2). Differentiation between G. camelum and G. martinicense from G. multipileum is evident through the presence of sessile basidiomata in the latter, as G. multipileum rarely exhibits this characteristic. The basidiomes of G. parvulum exhibit a range from sessile to stipitate, with a more frequent stipitate form akin to G. mizoramense. The basidiomata of G. mizoramense are pileate, stipitate, applanate, flabelliform, and devoid of any “growing zones,” contrasting with G. camelum. The pileal surface is smooth, laccate, radially rugose, slightly zonate with dark lines, and ranges from fully reddish brown to violet brown in G. parvulum. In contrast, it is small, soft, non-laccate and light camel brown in our new species. The upper pileus surface of G. mizoramense can be distinguished from G. camelum by its reddish brown (fresh) to liver-brown (dried) context, dark-brownish to dark reddish brown, with a white lower surface when fresh, as opposed to the light camel brown pileus and concolorous context observed in our new species.

Concentric growth zones are present in both G. camelum and G. martinicense. The contextum is dark cinnamon brown in G. martinicense and light brown to brown in G. multipileum, while our new species exhibits a soft velvety light brown or camel brown contextum. G. parvulum possesses a light pale ochraceous context (Torres-Torres and Guzmán-Dávalos, 2012) with dark horny (Murrill, 1902) or carob brown (Steyaert, 1980) resinaceous streaks, differing from our new species. The context in G. parvulum occasionally displays scattered yellow spots and a thin yellow line just below the crust, features absent in our new species. A uniform ochraceous or cinnamon context is characteristic of G. mizoramense.

In G. parvulum, the margins are slightly lobulated, ranging from white to pale yellow or grayish-yellow to yellowish orange, contrasting with the white and non-lobulated margins of G. camelum. Our species features a wide yellowish-brown porous surface, contrasting the white, yellowish white, or sun yellow surface in actively growing G. parvulum and the yellowish brown to brownish orange surface in dried pore surfaces.

Cuticular cells are cylindrical to slightly clavate, averaging 50 μm in length, while those in our new species are palissade club-shaped, clavate, pale yellow to yellow, smooth, double-walled, with few two-to three-septate or few multi-septate cells, and smaller in size (32.5–52.7 μm × 10.2–12.4 μm).

Ganoderma camelum has smaller basidiospores (4.7–5.2 μm × 2.3–3.6 μm), subglobose to ellipsoid, smooth, bitunicate, and lacks laterally pointed and inter-walled pillars. In contrast, G. parvulum features larger spores (8.1 μm × 5.9 μm), free to subfree very thin pillars. The basidiospores of G. mizoramense are brown, ellipsoid with a truncate base, verruculose, and larger (11.10 μm × 7.6 μm) than those of our new species. The spore size in G. multipileum and G. martinicense is larger (7.3–) 8.0–11.5 (−12.2) × (5.3–) 5.5–7.8 (−8.3) μm and (8–)8.8–10.5(−11.3) × (5–)5.5–7(−7.2) μm, respectively, while our new species exhibits smaller spores.

Four Trichoderma species, including T. cremeum, T. longipile, T. citrinoviride, and T. atroviride, were found to be capable of oxidizing guaiacol. Ganoderma exhibited the darkest maroon zone on the laccase detection plate, whereas Trichoderma demonstrated the highest laccase activity, as observed in Supplementary Figure S1A1,A2. Among the tested species, T. atroviride was identified as having the greatest laccase secretion potential, while T. citrinoviride presented the lowest. Consequently, T. atroviride secreted the highest amount of laccase, as depicted in Supplementary Figure S1B1,B2. On MEA media, T. atroviride produced pale green spores, with the prevalence of green indicating its dominant zones of operation. The growth rate within the Ganoderma zonal area was slower.

This study identified the most potent Trichoderma candidates under optimal conditions and compared their laccase activity to that of Ganoderma camelum, which had a laccase activity of 8.3 U/mL. Trichoderma atroviride was found to exhibit a laccase activity of 2.62 U/mL, establishing it as the most promising candidate for laccase production. The secondary productive species, T. cremeum, T. longipile, and T. citrinoviride, yielded laccase concentrations of 1.98 U/mL, 1.84 U/mL, and 1.65 U/mL, respectively (Table 3). The most robust candidate, T. atroviride, was selected for subsequent studies.

The purification of laccase from Trichoderma atroviride and Ganoderma camelum was carried out using 60% ammonium sulfate precipitation. After partial purification, the molecular weights of the laccase were determined using SDS-PAGE. Standard protein markers were employed to estimate the purified laccase, with the band positions post-staining aiding in this quantification. The molecular weights were approximately 57.0 kDa for T. atroviride and 62.0 kDa for G. camelum (Supplementary Figure S2).

The sequence of events commencing with reconnaissance culminates in the penetration of fungal pathogens, ultimately leading to host mortality. The secretion of laccase by G. camelum facilitated rapid mycelial advancement toward Trichoderma. In a competitive interaction, both species secreted laccase within their immediate environment, collaboratively executing the task. Ganoderma mycelium established physical contact with Trichoderma hyphae within the laccase oxidation zone, subsequently penetrating the lumen of the Trichoderma hyphae and assimilating its contents. Both Ganoderma and Trichoderma species released laccase enzymes or vied for space and nutrients during the antagonistic interaction.

Microscopic examination of the interaction between pathogenic G. camelum and T. atroviride revealed hyphal growth, followed by coiling, entanglement, and hooking around G. camelum. A change in medium color from white mycelium to purple indicated laccase secretion.

In vitro plate studies demonstrated that Ganoderma inhibited the growth of all Trichoderma species, which may be attributed to the higher molecular weight of laccase. An inhibition level exceeding 70%, as evaluated by the PIRG equation, was considered indicative of a species’ potential against another.

All Trichoderma species produced laccase at varying rates and inhibited G. camelum to different extents (Table 3). Trichoderma atroviride exhibited the highest PIRG score of 28.7%, which was statistically significant compared to other species. Regarding laccase production, five species were found to exhibit the highest protective effect against Ganoderma: T. atroviride, T. viride, T. virens, T. pseudokoningii, and T. cremeum, as determined by the PIRG evaluation.

Among these, T. atroviride secreted the highest amount of laccase, followed by T. cremeum, T. longipile, and T. citrinoviride. T. atroviride also effectively inhibited G. camelum, while T. cremeum, T. longipile, and T. citrinoviride also displayed rapid growth and purplish pigmentation after 5 days of incubation. Over a 10-day observation period, T. citrinoviride exhibited the smallest inhibitory zone against G. camelum development (76.3%) (Figures 5A–J).

The antagonistic interaction between Ganoderma and Trichoderma was confirmed through in vitro plate analysis, which revealed that Ganoderma suppressed the growth of all Trichoderma species. As assessed by the PIRG equation, an inhibition level above 70% was deemed indicative of a species’ antagonistic potential.

Considerable alterations in hyphal morphology were detected in Ganoderma mycelia upon exposure to Trichoderma, in contrast to the control group.

Figure 4 reveals a robust and well-developed Ganoderma mycelium, characterized by its healthy, compact, and highly branched structure. In response, the mycelium of G. camelum adopted a highly branched structure to mitigate the growth of Trichoderma. This adaptation was facilitated by producing a copious amount of laccase, thereby maximizing its defensive potential.

The laccase zone expanded across the entire surface of the Petri plates; however, only a limited number of hyphal strands were colonized and obscured by Trichoderma spores.

The hyphal structure of T. atroviride showed signs of disruption, aggregation, shriveling, loss, flattening, and altered appearance. Mycelial damage was attributed to intense resource competition, ultimately inhibiting Trichoderma growth.

The attachment of Trichoderma spores to the fungal hyphae indicated a mycoparasitic interaction. Trichoderma sp. was observed to identify and encircle adjacent fungal hyphae, forming haustoria to penetrate cell walls as a defensive strategy (Supplementary Figure S4).

Airborne basidiospores of G. camelum are released through appropriate openings in the injured area of woody tissue near the ground. The basal region of the tree is prone to damage due to the moisture present in the soil.

Structural roots in the soil are damaged by basidiospores and gradually colonized, leading to tree death over several years. Structural roots anchor the tree in the soil, while fine feeder roots absorb moisture and nutrients daily.

Once structural roots are damaged, stability is at risk due to colonization by Ganoderma species. No effective prevention or control measures are available to overcome BSR disease until the tree’s demise. However, the Trichoderma species have also proved ineffective except for removing and replacing soil and trees. In this study, infected trees were compared with healthy ones as control. The vigor of Pinus wallichiana declined, resulting in structural weakness, slow growth, yellowing and shrinking leaves, susceptibility to wind damage, and, eventually, branch dieback.

Once colonized, the wood block becomes saturated with water, resulting in a fibrous, tender, porous, crumbly, and flaky texture. The wood becomes discolored, primarily in white hues with occasional yellowish tones. The weight of the block was measured every 20 days, decreasing from 98.2 g initially to 97.5 g after 40 days, 97.1 g after 60 days, and finally 95.3 g after 120 days (Figure 6). The infected areas develop a pale, patchy appearance due to the presence of mycelium.

The woody stem becomes discolored, forming white patches due to laccase-induced degradation.

The mycelium growing within the softened wood secretes enzymes that degrade the cell wall components for energy and nourishment. Consequently, the wood undergoes whitening and bleaching due to oxidation by laccase, leading to lignin degradation.

Principal component analysis (PCA) was conducted, yielding two variables that accounted for 100% of the system’s variability. All parameters exerted a very strong influence on the variability of the system (Figure 7A). The PIRG rate and laccase activity parameters were strongly and positively correlated.

The PCA analysis indicated that positive values of the first principal component (PC1) described the correlation and influence of the PIRG rate and laccase parameters, accounting for 98.59% of the variance. The conducted PCA analysis demonstrated that positive values of the first main component, PC1, described the types of fungi at 98.59%. Positive values of the first principal component, PC1, disclosed the Ganoderma type, and negative values depicted the Trichoderma type. The PCA analysis (Figure 7B) also revealed that the PIRG rate and laccase parameters were described by Ganoderma camelum.

The heat map visually represents data, highlighting value variations through color contrasts. This graphical depiction serves as an effective means of illustrating the matrix’s values through a spectrum of colors. As detailed in Table 4, the correlation matrix shown the correlation coefficients for every pair of variables. In this study, the row and column labels consist of the “names of the variables” and the “numerical values of the calculated correlation coefficients,” which are explicitly listed within the table. The correlation coefficient, ranging from -1 to 1, indicated the strength of the linear relationship between the variables. The greater the absolute value, the more pronounced the relationship. Moreover, the sign of the correlation coefficient indicates whether the relationship between the studied variables is positive or negative.