CmWRKY6–1–CmWRKY15-like transcriptional cascade negatively regulates the resistance to fusarium oxysporum infection in Chrysanthemum morifolium

Abstract Chrysanthemum Fusarium wilt is a soil-borne disease that causes serious economic losses to the chrysanthemum industry. However, the molecular mechanism underlying the response of chrysanthemum WRKY to Fusarium oxysporum infection remains largely unknown. In this study, we isolated CmWRKY6–1 from chrysanthemum ‘Jinba’ and identified it as a transcriptional repressor localized in the nucleus via subcellular localization and transcriptional activation assays. We found that CmWRKY6–1 negatively regulated resistance to F. oxysporum and affected reactive oxygen species (ROS) and salicylic acid (SA) pathways using transgenic experiments and transcriptomic analysis. Moreover, CmWRKY6–1 bound to the W-box element on the CmWRKY15-like promoter and inhibited its expression. Additionally, we observed that CmWRKY15-like silencing in chrysanthemum reduced its resistance to F. oxysporum via transgenic experiments. In conclusion, we revealed the mechanism underlying the CmWRKY6–1–CmWRKY15-like cascade response to F. oxysporum infection in chrysanthemum and demonstrated that CmWRKY6–1 and CmWRKY15-like regulates the immune system.


Introduction
Fusarium wilt is a common plant disease caused by Fusarium oxysporum. F. oxysporum is the main pathogen invading the roots of plants, causing root rot, vascular blockage, leaf yellowing, and wilting, eventually leading to plant death due to a lack of water supply to the plant [1][2][3]. Fusarium wilt causes serious economic losses to the industry, necessitating the development of new disease-resistant varieties to control this disease. Plants have developed specific innate immune mechanisms during evolution to combat such pathogen infections. One such defense mechanism is the pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), which is activated by the specific recognition of PAMPs by pattern recognition receptors on the plant cell surface [4]. However, some pathogens can evade PTI by counteracting these factors, in which case, the plants activate the second mechanism of defense, effector-triggered immunity (ETI), by secreting resistance proteins specifically recognizing the pathogen effectors [5]. ETI is more rapid and intense than PTI. A complex immune network is involved in PTI and ETI for efficient immune regulation in plants [6]. Transcription factors affect the downstream immune system by identifying and regulating target genes.
WRKY is a plant-specific transcription factor that regulates plant biotic and abiotic stress responses and plays important reg-ulatory roles in the plant immune response [7][8][9]. WRKY contains a WRKYGQK sequence at its N-terminal end and a zinc finger structure at its C-terminal end. Zinc finger structure is divided into C-X 4-5 -C-X 22-23 -H-X 1 -H and C-X 7 -C-X 23 -H-X 1 -C. WRKY family is divided into three major groups: I, II, and III [10]. Group I contains two WRKYGOK sequences and one C-X 4-5 -C-X 22-23 -H-X 1 -H structure, group II contains one WRKYGQK sequence and one C-X 4-5 -C-X 22-23 -H-X 1 -H structure, and group III contains one WRKYGQK sequence and one C-X 7 -C-X 23 -H-X 1 -C structure [11,12]. WRKY plays an important role in the plant immune response. AtWRKY28/75 and AtWRKY33 increase the plant resistance to Sclerotinia sclerotiorum and Botrytis cinerea, respectively [13,14]. OsWRKY45 and OsWRKY53 increase the plant resistance to the rice blast pathogen, Magnaporthe grisea [15,16]. In addition, WRKY can specifically recognize and bind to W-box elements (TTGACC/T). W-box elements are present in the promoters of many plant defence-related genes, and WRKY can regulate plant defence-related genes, thereby affecting the plant defense systems [17]. OscWRKY1 positively regulates the expression levels of the phenylpropanoid pathway genes by binding to W-box elements in PAL and C4H promoters, thereby enhancing the plant resistance to bacteria [18]. Phosphorylated CaWRKY64 binds to the W-box element in the CaEDS1 promoter and enhances the resistance to F. oxysporum in chickpeas [19]. Soybean GmWRKY31 binds to the W-box element in the downstream GmSAGT1 pro-moter, increasing GmSAGT1 transcription and enhancing plant resistance to soybean downy mildew [20].
Reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ), superoxide anions (O2-), and hydroxyl radicals (-OH), are produced by plants in response to adversity [21]. ROS can act as second messengers to trigger the plant defense response. However, excessive accumulation of ROS can cause serious damage to proteins and nucleic acids, eventually leading to programmed cell death in plants [22]. To maintain the balance of ROS, plants have specific enzymes, such as peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO), glutathione reductase, and ascorbate peroxidase [23]. POD, CAT, and PPO can be used as physiological indices to evaluate the plant resistance to pathogens [24,25]. WRKY is a key transcription factor regulating ROS production. WRKY8 activates RBOHB expression and induces a hypersensitivity response in tobacco [26]. WRKY1 activates long non-coding RNA (lncRNA)-33 732 to induce RBOH expression, promote H 2 O 2 production, and improve tomato resistance to Phytophthora infestans [27].
Salicylic acid (SA) has been suggested to be an ROS homeostasis regulator in plants. SA is a phenolic compound with antioxidant properties that plays an important regulatory role in the immune response of plants [28,29]. SA increases the levels of glutathione, inducing the elimination of ROS in plants [30,31]. In addition, WRKY can participate in plant immunity by regulating the SA pathway. Inhibition of SA signaling by CaWRKY70 increases the susceptibility of chickpeas to F. oxysporum [32]. We previously reported that CmWRKY8-1 affects resistance to F. oxysporum by regulating the genes involved in the SA synthesis pathway in chrysanthemum [33].
Chrysanthemum is extensively cultivated in China and loved by the general population. However, Fusarium wilt causes serious damage to chrysanthemum plants. At present, there are more and more studies on the mining of chrysanthemum resistance functional genes [34,35]. Identifying the molecular mechanisms underlying the chrysanthemum response to F. oxysporum infection is necessary to manage this pathogen. Song reported that CmWRKY6 is involved in F. oxysporum infection, suggesting its involvement in the response of chrysanthemum to F. oxysporum infection [36]. In this study, we cloned CmWRKY6-1 and found that it negatively regulated resistance to F. oxysporum and affected ROS and SA pathways. We also identified a regulatory relationship between CmWRKY6-1 and CmWRKY15-like. Overall, we revealed a novel pathway for chrysanthemum response to F. oxysporum infection that may be used as a basis for future disease resistance studies of chrysanthemum WRKYs.

Sequence analysis of CmWRKY6-1
In previous experiments, Song found that CmWRKY6 (KC615360) responds to F. oxysporum infection [36]. To identify the mechanism by which CmWRKY6 responds to F. oxysporum infection, we cloned and isolated a gene sequence with a 699 bp open reading frame (ORF) from 'Jinba' variety (Table S1, see online supplementary material). Homologous sequence analysis revealed that the protein sequence contained a WRKY structural domain with the zinc finger structure, C-X 5 -C-X 23 -H-X 1 -H, belonging to WRKY family II. Notably, it has only one amino acid difference from the CmWRKY6 protein sequence; therefore, we named it CmWRKY6-1 (Fig. 1A). Phylogenetic tree revealed that CmWRKY6-1 has the highest affinity for Tanacetum cinerariifolium TcWRKY21 (Fig. 1B).
Next, we transformed pGBKT7-CmWRKY6-1 in a yeast twohybrid (Y2H). We found that transformed with the negative controls pGBKT7 and pGBKT7-CmWRKY6-1 did not grow normally on SD/Ade/His-deficient medium (Fig. 1D). Furthermore, we used chrysanthemum protoplasts to conduct transcriptional activity analysis. Luciferase (LUC) f luorescence signal intensity of CmWRKY6-1 was significantly lower than that of the positive control (Fig. 1E). These results indicate that CmWRKY6-1 does not exhibit any transcriptional activity.
To clarity the expression patterns of CmWRKY6-1 in different chrysanthemum tissues during growth, the terminal buds, stems, leaves, and roots of 'Jinba' were sampled and analysed via qRT-PCR. Relative expression levels of CmWRKY6-1 were the highest in buds, followed by the roots (Fig. 1F).

CmWRKY6-1 negatively regulates the resistance of 'Jinba' to F. oxysporum
To investigate the expression patterns of CmWRKY6-1 in response to F. oxysporum infection, we performed qRT-PCR assays on the roots of chrysanthemum 'Jinba' varieties after inoculation with F. oxysporum. Expression levels of CmWRKY6-1 were significantly more downregulated in the experimental group than in the control group at 3 h. Moreover, expression levels of CmWRKY6-1 were significantly lower in the experimental group than in the control group within 120 h ( Fig. 2A).
To further determine the functions of CmWRKY6-1 in chrysanthemum, we transfected the pORE-R4-CmWRKY6-1 plasmid into 'Jinba' and obtained two CmWRKY6-1 overexpression lines, OX-#1 and OX-#2 (Fig. S1, see online supplementary material). qRT-PCR revealed that the expression levels of CmWRKY6-1 in the overexpression lines were 3.7-and 4.4-fold higher than those in the wild-type (WT), respectively (Fig. 2B). As CmWRKY6-1 did not exhibit any transcriptional activation activity, we constructed the FVuv-R4-CmWRKY6-1 vector by fusing CmWRKY6-1 with VP64 to convert CmWRKY6-1 into a transcriptional activator [33]. The vector was then transferred into 'Jinba' to obtain two CmWRKY6-1 interfering lines, Fvuv-#1 and Fvuv-#2 (Fig. S2, see online supplementary material). Expression levels of CmWRKY6-1 were 7.3and 6.3-fold higher in the interfering lines than in the WT, respectively (Fig. 2C). Next, we simultaneously inoculated the WT and CmWRKY6-1 transgenic lines with F. oxysporum. Basal leaf wilting was more severe in OX lines than in WT and Fvuv lines ( Fig. 2D; Fig. S3, see online supplementary material). In contrast, the basal leaves of Fvuv lines exhibited only a slightly wilted phenotype and were significantly less affected than WT and OX lines ( Fig. 2D; Fig. S3, see online supplementary material). We also calculated the disease severity index (DSI) of each line and found that OX lines had higher DSI than WT, whereas Fvuv lines had lower DSI than WT (Fig. 2E). These results suggest that CmWRKY6-1 negatively regulates resistance to F. oxysporum in chrysanthemum.

Transcriptome analysis of CmWRKY6-1 overexpression line
To further determine the mechanism underlying the CmWRKY6-1 response to F. oxysporum infection, we conducted transcriptome analyses of root samples from OX-#1 and WT lines 0, 3, and 72 h after inoculation (control group: WT-0 h, WT-3 h, and WT-72 h;

CmWRKY6-1 negatively regulates the ability to eliminate ROS in chrysanthemum
We further conducted Gene Ontology (GO) analysis of DEGs at various time points and found that redox processes, including responses to ROS, were significantly enriched (Fig. 3A). We hypothesized that CmWRKY6-1 regulates the redox pathway.
To verify this hypothesis, we inoculated transgenic and WT lines with F. oxysporum and stained the infected basal leaves with nitrotetrazolium blue chloride (NBT) and diaminobenzidine (DAB). Levels of ROS were higher in the leaves of the inoculated CmWRKY6-1-OX lines than in those of the WT and FVuv-CmWRKY6-1 lines, whereas the opposite trend was observed in the FVuv-CmWRKY6-1 lines (Fig. 3B and C). We also measured the activities of CAT, POD, and PPO-related redox pathway enzymes in each line. CmWRKY6-1-OX lines had lower levels of related enzyme activities than the WT, whereas the opposite trend was observed in the FVuv-CmWRKY6-1 lines (Fig. 3D). These results indicate that CmWRKY6-1 negatively regulates the ability to eliminate ROS in chrysanthemum.

CmWRKY6-1 negatively regulates the synthesis of endogenous SA in chrysanthemum
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs revealed a significant enrichment in the plant hormone signal transduction pathway (Fig. 4A). We previously reported that chrysanthemum WRKY responds to F. oxysporum infection by regulating the SA pathway [33]. To determine whether CmWRKY6-1 is involved in the SA pathway, we further analysed the changes in the expression levels of key genes related to the SA pathway using the transcriptome data after plant inoculation.  (Fig. 4B). To validate the RNA-seq results, we measured the expression levels of these genes via qRT-PCR. qRT-PCR results were consistent with those of RNA-seq (Fig. 4C). Furthermore, we determined the levels of endogenous SA after F. oxysporum inoculation. Indeed, CmWRKY6-1-OX lines had lower endogenous SA levels than the WT, whereas the opposite trend was observed in the FVuv-CmWRKY6-1 lines (Fig. 4D). In conclusion, we demonstrate that CmWRKY6-1 negatively regulates the synthesis of endogenous SA in chrysanthemum.

CmWRKY15-like
In a previous report, CmWRKY15-1 positively regulates chrysanthemum resistance to Puccinia horiana Henn via the ROS and SA pathways [37,38]. In the above experiments, we also found that CmWRKY6-1 can regulate the ROS and SA pathways. We hypothesized that there is a direct or indirect relationship between CmWRKY6-1 and CmWRKY15-1 in the ROS and SA pathways to regulate downstream defense systems.
To verify this hypothesis, we designed primers (Table S3, see online supplementary material) and cloned an ORF of 807 bp (Table S1, see online supplementary material). We then compared the obtained sequence with the CmWRKY15 sequence and identified only three amino acid differences between the two, accounting for 97% similarity of the obtained sequence to CmWRKY15-1 sequence (Fig. S6, see online supplementary material). Moreover, the obtained sequence was localized in the nucleus (Fig. 5A), and hence, named CmWRKY15-like. Notably, we found that the expression levels of CmWRKY6-1 and CmWRKY15-like were negatively correlated using transcriptomic data (Fig. 5B). The relative expression of CmWRKY15-like was lower in CmWRKY6-1-OX lines than in CmWRKY6-1-FVuv lines (Fig. S7, see online supplementary material). We also cloned the promoter fragment of CmWRKY15-like with a length of 2048 bp (Table S4, see online supplementary material). Using the yeast single-hybrid assay and EMSA, we verified that CmWRKY6-1 bound to the W-box element in the CmWRKY15-like promoter ( Fig. 5C and D). In addition, CmWRKY6-1 inhibited CmWRKY15like expression in the dual-luciferase system ( Fig. 5E and F). These results indicate that CmWRKY6-1 directly inhibits CmWRKY15like expression.

CmWRKY15-like silencing negatively regulates 'Jinba' resistance to F. oxysporum
After determining that CmWRKY6-1 directly inhibited the downstream CmWRKY15-like expression, we investigated the specific functions of CmWRKY15-like in response to F. oxysporum infection. We obtained CmWRKY15-like interfering lines (RNAi-#1 and RNAi-#2; Fig. S8, see online supplementary material) and inoculated them with F. oxysporum to observe the resultant phenotype. CmWRKY15-like interfering lines exhibited a higher degree of pathogenic phenotype than the WT (Fig. 6A). We also calculated DSI of each line and found that RNAi lines had higher DSI than WT (Fig. 6B). In addition, endogenous SA levels of the interfering lines were lower than those of the WT (Fig. 6C). These results indicate that CmWRKY15-like silencing negatively regulates chrysanthemum resistance to F. oxysporum.

CmWRKY6-1 negatively regulates chrysanthemum resistance to F. oxysporum and affected ROS and SA pathways
WRKY is an important transcription factor for plant resistance that regulates defense responses against pathogens in various plants, such as rice, apple, and grapevine plants [39][40][41]. In our previous study on chrysanthemum Fusarium wilt, we reported that CmWRKY8-1 affects resistance to F. oxysporum by regulating SA-related synthetic genes, such as ICS1, PAL, PR1, PR2, and PR5 [33]. In this study, we identified CmWRKY6-1 as a nuclear transcriptional repressor. Through transgene functional validation, we confirmed that CmWRKY6-1 negatively regulated chrysanthemum resistance to F. oxysporum.
Comparison of the CmWRKY6-1 overexpression and WT lines transcriptome data revealed that DEGs in the ROS and SA pathways were significantly enriched. Although the PTI and ETI responses belong to two levels of defense, they can jointly regulate ROS and SA signaling pathways to induce downstream defense responses [42]. ROS are metabolites of oxygen and its derivatives. Plants can rapidly produce ROS under abiotic and biotic stresses, which can increase their susceptibility to necrotrophic pathogens as well as their resistance to live trophic pathogens [43]. With the increase in stress degree and time, the ability of plants to eliminate ROS is seriously affected, and the basic metabolic pathways of plants are damaged [44]. SA is an important plant defense hormone that has been reported in Arabidopsis thaliana and tobacco [45]. SA was reported to respond to F. oxysporum infection in a previous study on chrysanthemum Fusarium wilt [46]. We confirmed that CmWRKY6-1 negatively regulated ROS scavenging using NBT and DAB staining as well as enzyme activity assays. We further demonstrated that CmWRKY6-1 inhibited SA synthesis by reducing the expression levels of SA pathway-related genes. In conclusion, CmWRKY6-1 inhibited ROS elimination and endogenous SA synthesis.

CmWRKY15-like
WRKY family regulates gene expression by recognizing and binding to W-box elements [47]. Chrysanthemum CmWRKY15-1 binds to the W-box element in CmNPR1 and interacts with CmNPR1 to improve its resistance to white rust [38]. In addition, WRKYs have been suggested to have mutual regulatory mechanism. When infected with F. oxysporum, chickpea CaWRKY40 binds to the W-box element in the CaWRKY33 promoter, enhancing defense against pathogens [48]. Pepper CaWRKY40 is both selfregulated and regulated by other WRKYs [49]. We have noticed that both CmWRKY15-1 and CmWRKY6-1 can regulate the ROS and SA pathways in chrysanthemum [37,38]. So, we hypothesized that there is a direct or indirect relationship between CmWRKY6-1 and CmWRKY15-1 in the ROS and SA pathways. We undertook the experiment to support the hypothesis. In this study, we obtained an additional copy of CmWRKY15-1, CmWRKY15-like, that exhibited 97% similarity with it. CmWRKY6-1 inhibited the expression of CmWRKY15-like by binding to the Wbox element in the CmWRKY15-like promoter. When CmWRKY15like was silenced, chrysanthemum plant was susceptible to F. oxysporum, indicating that CmWRKY15-like is functionally important for chrysanthemum to resist F. oxysporum infection. Our results revealed the regulatory relationship between CmWKRKY6-1 and CmWRKY15-like, providing a new theoretical basis for the disease resistance regulatory network among WRKYs.
regulates resistance to white rust by increasing ROS scavenging and SA levels [37,38]. Therefore, we proposed the hypothesis that CmWRKY6-1-CmWRKY15-like cascades reduces resistance to F. oxysporum in chrysanthemum by regulating the ROS and SA pathways (Fig. 7). This is currently under investigation in our lab.

Plant materials
We used the chrysanthemum cultivar 'Jinba' and CmWRKY6-1 transgenic lines provided by the Chrysanthemum Germplasm Resource Preserving Center, Nanjing Agricultural University (Nan-

Inoculation of F. oxysporum
F. oxysporum was isolated from the chrysanthemum cultivar, 'Jinba' [33]. Fungal cakes were inoculated into potato dextrose agar culture, incubated at 28 • C for six days, split into 0.7-cm cakes, inoculated in 500 mL of PDB, and incubated for five days at 28 • C and 180 rpm. Next, the chrysanthemum roots were lightly wounded with scissors and immersed in a spore suspension at a concentration of 10 7 CFU/mL for 30 min. When chrysanthemums were set, they were inoculated with 1 × 10 7 spores per gram of substrate soil. Finally, the chrysanthemums were cultured in a phototemperature chamber under 16/8 h (light/dark) cycle at 28 • C and 80% humidity.

Isolation and sequence analysis
RNA from 'Jinba' was extracted and reverse transcribed into cDNA. ORF sequences of CmWRKY6-1 and CmWRKY15-like were amplified using cDNA as the template and the primers CmWRKY6-1-F/R and CmWRKY15-like-F/R (Table S3, see online supplementary material). We downloaded the homologous sequence of CmWRKY6-1 from GenBank (https://www.ncbi.nlm. nih) and constructed a phylogenetic tree by the neighbor-joining method using MEGA X with 1000 bootstrap replications. We used the DNAMAN software to perform homologous sequence comparisons.

Subcellular localization
pORE-R4-CmWRKY6-1 and pORE-R4-CmWRKY15-like vectors were constructed. 35S::D53-RFP, pORE-R4-CmWRKY6-1 and pORE-R4-CmWRKY15-like were transfected into Agrobacterium tumefaciens strain, GV3101. They were then injected into tobacco leaves via transient transformation and cultured for one day in the dark and two days in the light before observing the f luorescence signal under a confocal microscope.

Transactivation assays
Construction of pGBKT7-CmWRKY6-1. pGBKT7-CmWRKY6-1, positive control pCL1, and negative control pGBKT7 were transferred into the Y2H yeast receptor state. The positive control yeast was coated with SD/Leu-deficient media, and the negative control and pGBKT7-CmWRKY6-1 yeast were coated with SD/Trp-deficient media. After 4-5 d at 28 • C, colonies were picked and spotted on SD/Ade-His-deficient media with or without X-α-gal. Yeast growth was observed by capturing photographs after 3-5 d.
Chrysanthemum protoplasts were extracted from the leaves of chrysanthemum histoculture seedlings grown for approximately one month [53]. The luminescence of LUC was measured using a GLOMAX ® -20/20 instrument.

qRT-PCR
Quantitative primers were designed using the Primer Premier software (5.0) ( Table S3, see online supplementary material). CmEF1α was selected as the reference gene [54,55]. Each sample was replicated thrice, and the results were calculated using the 2 -ct method [56].

Chrysanthemum transformation and phenotype analysis
pORE-R4-CmWRKY6-1, FVuv-R4-CmWRKY6-1, and amiR-CmWRKY15like vectors were constructed, transferred into A. tumefaciens EHA105, and transformed into chrysanthemum using a previously described method [57,58]. DNA of the transgenic lines was extracted and identified at the DNA level using vector and gene primers (Table S3, see online supplementary material). RNA from the transgenic lines was extracted and reverse transcribed to cDNA, and the expression level was determined using quantitative primers for the gene (Table S3, see online  supplementary material).
'Jinba', which had been cut for 40 d, was inoculated with F. oxysporum, and the DSI (Table S5, see online supplementary material) was recorded when obvious morbidity phenotype was observed [33].

ROS and SA analysis of chrysanthemum
Roots and basal leaves of plants were collected after infection. Leaves were stained with DAB and NBT. Brief ly, the diseased leaves were immersed in DAB solution for 12 h in the dark, decolored with alcohol, and finally photographed and stored. The diseased leaves were soaked in NBT solution for 12 h, decolorized with alcohol, photographed, and stored. Enzymatic activity were measured using the corresponding kits (Comin, Suzhou, China). We also determined the endogenous SA levels in the roots using the SA kit (Lengton Bioscience Co, Shanghai, China).

Yeast one-hybrid assay
'Jinba' DNA was extracted and primers (Table S3, see online supplementary material) were designed to clone the CmWRKY15like promoter sequence. CmWRKY15-like promoter sequence was inserted into the pHIS2 vector. pGADT7-CmWRKY6-1 vector was constructed. pGADT7-CmWRKY6-1 and pHis2-CmWRKY15-like pro vectors were co-transformed into the yeast strain, Y187. pGADT7 and pHis2-CmWRKY15-like pro co-transformed yeast were the negative controls. After transformation, the yeast cells were cultured in SD/Trip/His/Leu-deficient medium. After three days, the yeast were picked and transferred to SD/Trip/His/Leu-deficient medium with 90 mM and without 3-amino-1,2,4-triazole, incubated for three days, and photographed for observation.

EMSA
pGEX-5X-1-CmWRKY6-1 vector was constructed. Cells were incubated at 16 • C. Probes and mutation probe primers (Table S3, see online supplementary material) were designed and the primer pairs were labeled. CmWRKY6-1 binding to CmWRKY15-like pro was validated using the EMSA kit [50].

Luciferase assays
CmWRKY15-like promoter was inserted into the pGreenII 0800-Luc vector and transformed into A. tumefaciens. pORE-R4-CmWRKY6-1 and pGreenII 0800-Luc-CmWRKY15-like pro were transiently transfected into the tobacco leaves. pORE-R4 empty and pGreenII 0800-Luc-CmWRKY15-like pro were the negative controls. Cells were cultured in the dark for 24 h, then in the light for 48 h. Leaves were sprayed with D-f luorescein sodium salt and observed using a Tanon 5200 multi-imaging device (Tanon, Shanghai, China).

Statistical analyses
Data analysis were conducted using SPSS. Analysis of variance and t-tests were used to analyse all data to determine the significant differences.