Bacillus licheniformis strain POT1 mediated polyphenol biosynthetic pathways genes activation and systemic resistance in potato plants against Alfalfa mosaic virus

Alfalfa mosaic virus (AMV) is a worldwide distributed virus that has a very wide host range and causes significant crop losses of many economically important crops, including potato (Solanum tuberosum L.). In this study, the antiviral activity of Bacillus licheniformis strain POT1 against AMV on potato plants was evaluated. The dual foliar application of culture filtrate (CF), 24 h before and after AMV-inoculation, was the most effective treatment that showed 86.79% reduction of the viral accumulation level and improvement of different growth parameters. Moreover, HPLC analysis showed that a 20 polyphenolic compound was accumulated with a total amount of 7,218.86 and 1606.49 mg/kg in POT1-treated and non-treated plants, respectively. Additionally, the transcriptional analysis of thirteen genes controlling the phenylpropanoid, chlorogenic acid and flavonoid biosynthetic pathways revealed that most of the studied genes were induced after POT1 treatments. The stronger expression level of F3H, the key enzyme in flavonoid biosynthesis in plants, (588.133-fold) and AN2, anthocyanin 2 transcription factor, (97.005-fold) suggested that the accumulation flavonoid, especially anthocyanin, might play significant roles in plant defense against viral infection. Gas chromatography-mass spectrometry (GC-MS) analysis showed that pyrrolo[1,2-a]pyrazine-1,4-dione is the major compound in CF ethyl acetate extract, that is suggesting it acts as elicitor molecules for induction of systemic acquired resistance in potato plants. To our knowledge, this is the first study of biological control of AMV mediated by PGPR in potato plants.

www.nature.com/scientificreports/ isolates from different parts of the world with 99% and presented as a monophyletic group of these isolates (data not shown).
Effect on symptom development, AMV accumulation level and growth parameters. The symptoms of AMV including yellow blotching and bright mottling ended with clear visibility of calico symptoms on non-treated infected potato plant leaves (T2) were observed at 21 dpi (Fig. 1). The symptom appearance on POT1 treated plants 24 h before viral inoculation (T3) and POT1 treated plants before and after viral inoculation (T4) treatment was delayed approximately three and five days, respectively, compared to T2 treatment. Comparing to T1 plants, T2 plants exhibited the higher accumulation level of AMV (33.33-fold) in AMVinfected potato leaves. On the other hand, a significant decrease in the viral accumulation level in T3 (18.23-fold) and T4 (4.40-fold) treatment plants was observed (Fig. 2).
The data of the potato plant parameters from the greenhouse experiment showed significant reduction in tuber number, tuber weight, fresh weight and dry weight of potato plants that were infected with AMV (T2), recording 2.33 ± 0.58 g, 22.32 ± 1.09 g, 20.12 ± 1.07 g, and 2.46 ± 0.07 g, respectively, compared to control plants ( Table 2). The foliar applications of POT1 either T3 or T4 were significantly increased potato tuber numbers,   CF 24 h before inoculation of AMV, and T4 = plant treated with CF, 24 h before inoculation of AMV and 24 h after inoculation with AMV. Columns represent mean value from three biological replicates and bars indicate Standard Deviation (± SD). Significant differences between samples were determined by one-way ANOVA using CoStat software. Means were separated by Least Significant Difference (LSD) test at P ≤ 0.05 levels and indicated by small letters. Columns with the same letter means do not differ significantly.

Scientific RepoRtS
| (2020) 10:16120 | https://doi.org/10.1038/s41598-020-72676-2 www.nature.com/scientificreports/ tuber weights, fresh weights and dry weights in comparison to AMV-infected potato plants (T2). The T4 treatment accomplishes the adverse effects of the disease by increasing tuber number or weight with 4.33 ± 0.58 and 60.26 ± 2.23 g, respectively. Moreover, the fresh weight as well as the dry weight of the T4 treatment were significant greater than those of the other treatments at 70 dpi. No disease symptoms were observed on the non-infected plants.
transcriptional levels of polyphenol biosynthetic pathways-related genes. It is well known that, plant polyphenolic compounds, secondary metabolites, play important roles in plant growth and defense against different biotic and abiotic stresses. The transcriptional expression levels of thirteen genes encoding the essential enzymes regulating the polyphenol biosynthetic pathways were investigated at 21 dpi. The three-phenylpropanoid, chlorogenic and flavonoid biosynthetic pathways are the major route of polyphenol biosynthetic compounds.
the core phenylpropanoid biosynthetic pathway. The relative expression levels of the two genes, PAL and C4H, encoding the first two enzymes in phenylpropanoid biosynthetic pathway were evaluated. Compared to control (T1), a significant up-regulation of PAL with a relative expression of 2.928-and 2.462-fold change, no significant changes, was observed in T3 and T4 treatments, respectively (Fig. 3). Despite PAL exhibited downregulation with relative expression levels of 0.479-fold in T2 treatment, there was no significant change with control ( Fig. 3). On the other hand, C4H was induced and significantly up-regulated in T4 treatment plants with transcript level 6.291-fold increased than control (Fig. 3). Like PAL, C4H was also down-regulated in T2 treatment with a relative expression level of 0.703-fold, while it was quite equal in T3 treatment compared with the control plants, there was no significant change between T1, T2 and T3 (Fig. 3). Consequently, foliar spraying of potato plants with CF 24 h before inoculation with AMV (T3) induced expression of PAL only, while the dual treatments (T4), foliar spraying of potato plants with CF 24 h before inoculation with AMV and 24 h after inoculation, triggered the expression of both PAL and C4H, genes.
the chlorogenic acid biosynthetic pathway. The transcript levels of three genes (HCT, C3H and HQT) encoding three regulatory enzymes of chlorogenic acid biosynthesis were investigated. The significant up-regulation of HCT was observed in all treatments with relative expression levels of 1.203-, 3.732-and 2.441-fold change for T2, T3 and T4, respectively, compared to control (Fig. 3). The highest transcript level (8.111-fold) of C3H was observed in T4 treatment, followed by T3 and T2 with a relative transcript level of 7.06-and 6.453-fold change, respectively, compared to control (Fig. 3). These results showed that AMV infection induced expression of HCT and C3H in potato and the expression increased with POT1 treatments either before or after infection. On the other hand, the obtained data indicated that the HQT gene was not induced neither AMV infection nor POT1 treatments. The down-regulation with relative expression levels of 0.062-, 0.070-and 0.018-for T2, T3 and T4, respectively, was observed (Fig. 3).
The flavonoid biosynthetic pathway. Compared to control, a clear differentiation in transcriptional profiles of eight genes (CHS, CHI, F3H, FLS, DFR, F3′H, AN1, and AN2) encoding eight enzymes controlling the flavonoid biosynthesis pathway was observed (Fig. 3). For CHS expression, up-regulation with a significant relative expression level (2.084-fold) was observed in AMV-infected plants (T2) when compared with the control plants. However, POT1-treated plants before infection (T3) or before + after (T4) exhibited higher expression levels with 4.823-and 5.656-fold change, respectively (Fig. 3). Thus, the treatment with either AMV or POT1 can trigger CHS expression level. Concerning CHI, the dual CF treatment (T4) showed the best results of CHI gene expression. The expression level was induced only in T4 treat-plants with a significant relative expression level of 1.777-fold-chang, while T2 and T3 showed down-regulation with relative expression levels of 0.636-and 0.703-fold, respectively, lower than control (Fig. 3). Among tested genes, F3H was the highest induced gene in all treatments when compared with control ( Fig. 3). However, AMV-infected plant (T2) showed a high expression level (116.162-fold), the POT1-treated plants before 24  www.nature.com/scientificreports/ level of FLS gene was shutdown in all treatments in comparison with control (Fig. 3). The down-regulation with relative expression levels of 0.147-, 0.266-and 0.463-fold in T2, T3 and T4, respectively, compared with control were observed. It was noted that the expressions of DFR and F3′H were very similar to each other. Although T2 treated plants exhibited a reduction of DFR and F3′H by relative expression levels 0.712-and 0.750-fold, respectively, no significant changes were reported when compared with control ( Fig. 3). On the one hand, T3 treated plants showed up-regulation with significant transcript levels 5.314-and 7.963-fold change for DFR and F3′H, respectively. Additionally the dual POT1 treatment (T4) was more enhancer than T3, resulting in the highest expression levels of 18.635-and 20.440-fold increased than control for DFR and F3′H, respectively (Fig. 3).
Concerning AN1 gene, significant up-and down-regulations with relative expression levels of 1.866-and 0.162fold change were showed in T4 and T2 treated-plants when compared with control ( Fig. 3). For AN2 transcript level, significant up-regulation in the transcription levels in all treatments was observed when compared with control. The highest induction with a relative expression level (97.005-fold) was showed in T4 treatment, while T2 treated-plants exhibited expression level of 20.821-fold change than control (Fig. 3).
phytochemical constituents of the potato leaf extract. The

Discussion
Plant viruses are among the most important plant pathogens, as near half of the emerging epidemics have a viral etiology, causing problems of food security and they are responsible for huge losses of crop production 29,30 . Due to negative impacts on public health and environmental hazards, chemical treatments including pesticide or insecticide must be managed and controlled. Biological control using PGPR (one or more strains) is being www.nature.com/scientificreports/ considered as an alternative or a supplemental way of handling of plant diseases better than the chemical control in agriculture [31][32][33][34] . Consequently, searching and discovering new environmental eco-friendly biocontrol agents capable to control plant viral diseases are demand 8 . In the current study, the antiviral activity of Bacillus licheniformis strain POT1 against AMV on potato (Solanum tuberosum L.) plant was evaluated. Moreover, the transcriptional levels of thirteen genes involved in phenylpropanoid, chlorogenic acid and flavonoid biosynthetic pathways genes, as well as bioactive constituents of POT1 crude filtrate (CF), were analyzed. Additionally, HPLC analysis was used to compare the content of polyphenol compounds with the expression levels of biosynthetic pathway genes. To our knowledge, this is the first time to deal with the effect of PGPR against AMV in potato plants.
Under greenhouse experiments, the application of POT1 has significantly increased plant growth, yield, reduced disease severity, and virus accumulation compared to infected potato plants without any treatments. We have shown that inhibition of AMV infection can be generated by treating plants with bacterial culture filtrate either prior to or post-challenge with the virus. Our results showed that the up-regulation of almost genes triggered by bacterial filtrate resulted in increasing resistance to AMV via limiting viral accumulation, symptom severity, and growth parameters increasing. Many reports showed that the application of some Bacillus spp., Pseudomonas spp. and Streptomyces spp., improved plant growth and increased protection against viral infection [35][36][37][38] .
In the current study, the obtained results showed that potato plants infected with AMV (T2) were significantly reduced the tuber numbers and weight as well as fresh and dry weight when compared with the control plants (T1). However, T4 treatment was significantly enhanced and improved all evaluated growth parameters recording the highest values of fresh and dry weight when compared to the other treatments and significantly reduced the negative effects resulted from the viral infection.
The greenhouse experiment results confirmed the effective biocontrol activity of POT1 against AMV infection, which resulted in a considerable decrease in AMV concentration levels. Significant reductions in virus concentration by 45.29% and 86.79% for potato leaves of T3 and T4 treatments, respectively, were showed when compared to T2 treatment. These results suggest that CF of POT1 contains secondary metabolites that can play a notable role in SAR. In this context, the foliar application of Streptomyces spp. CF exhibited a significant reduction of PVY in potato 39 . Thus, POT1 activates induced systemic resistance (ISR) of potato plants against AMV infection. ISR using PGPR showed promising results against plant viruses such as tomato mottle virus, CMV, and PVY in tomato 40 cucumber 41-43 and potato 44,45 , respectively. Besides the activation of some defense genes, ISR enhanced the production peroxidase, antioxidant protective enzyme and secondary metabolites 46,47 . Among the secondary metabolites, polyphenolic compounds play vital roles in plant growth and resistance against different biotic and abiotic stresses 19 . Routes to the major classes of polyphenol compounds involve three pathways (i) phenylpropanoid pathway, (ii) chlorogenic acid pathway and (iii) flavonoid pathway 27 .
The phenylpropanoid pathway started with the conversion of L-phenylalanine by PAL to cinnamic acid then to p-coumaric acid by cinnamic acid 4-hydroxylase (C4H) and ended by the formation of the main intermediate   56,57 . Consequently, the induction of PAL and C4H transcripts in T3 and T4 treatment suggesting that POT1 is a good elicitor activated ISR that is associated with SA and secondary metabolites, precursors of chlorogenic acid and flavonoids, biosynthesis in potato tissues. Chlorogenic acid, which is the ester of caffeic acid and quinic acid, is one of the polyphenol compounds, phenolic acids, that improving plant disease resistance through inhibiting pathogens 58,59 . The chlorogenic acid pathway started with the conversion of p-coumaroyl CoA to shikimate through HCT catalyzing activity 60 . Following synthesis of p-coumaroyl shikimate by C3H, HCT catalyzes the transfer of it to the caffeoyl CoA 61 ended with chlorogenic acid through HQT activity 62 . In the present study, although HCT showed slightly induced after AMV infection in T2 treatment (1.203-fold), the overexpression with relative expression level 3.732-and 2.441-fold change was observed in T3 and T4 treatments, respectively. Likewise, the up-regulation of C3H with the transcriptional expression level of 6.453-, 7.061-and 8.111-fold was reported in T2, T3 and T4, respectively. HCT and C3H involved in lignin biosynthesis in the plant cell wall 56,63 . Thus, the induction of transcriptional expression of these genes shows their protective role against AMV and suggests that the potato plant can use the lignifications as one of its defense to resist the viral infection and movement. On the other hand, the decreasing of chlorogenic and caffeic acids contents upon AMV-inoculation (T1, T2 and T3) could reflect the downregulation of transcriptional expression levels of HQT for these treatments when compared to control. Tomato HQT overexpression was associated with increases in chlorogenic acid content and versa versa 62 . Based on current results, whether HQT suppression and HCT and C3H induction, AMV-infected potato plant was associated with decreasing the content of chlorogenic acid. We can assume that AMV could not make complete suppression of chlorogenic acid biosynthesis and had a suppression effect on HQT rather than HCT and C3H. Moreover, the ISR activated by POT1 may be correlated with inducing and increasing cell wall lignifications.
The flavonoid pathway started with the conversion of p-coumaroyl CoA to naringenin chalcones, through CHS catalyzing activity, which can be transformed to naringenin by the action of CHI 27 . These are the first two steps of the flavonoid pathway and are strictly required for chalcones and dihydrochalcones production, which considered being the primary precursors and constituting the main intermediates for a large number of flavonoids The obtained results showed that F3H, the key enzyme in flavonoid biosynthesis in plants 66 , was the master expressed gene among flavonoid pathway genes with the highest expressions level 116.162-, 367.092-and 588.133-fold change than the control for T2, T3 and T4, respectively. Likewise, F3′H exhibited significant upregulation in both POT1 treatments with relative expression 7.963-and 20.440-fold increased than control. F3H converts hydroxylate naringenin to dihydroflavonol or dihydrokaempferol while F3′H, the primary enzymes responsible for the diversification of anthocyanins, transforms dihydrokaempferol into dihydroquercetin 67 [73][74][75] . DFR, a NADPH-dependent reducing enzyme, converts dihydroflavonols to leucoanthocyanidins which necessary for formation of anthocyanins in higher plants 76 . Quattrocchio et al. 77 reported that anthocyanin pathway is regulated at the DFR step in Petunia hybrid plants. Comparing to control in this study, both T3 and T4 treatments induced DFR transcripts with relative expression values of 5.314-and 18.635-fold change, respectively. Based on this data, we are suggesting that POT1 treatments stimulated the plant immune defense system to produce more anthocyanins-related compounds. AN1 and AN2 are two transcription factors involved in the regulation of anthocyanin biosynthesis 78 . Thus, a significant up-regulation (1.866-fold) of AN1 in T4 treatment could be induced biosynthesis of anthocyanin-related compounds. D' Amelia et al. 79 reported that AN1 expression is associated with high anthocyanin contents in leaves. On the other hand, the higher and stronger expression of AN2 with relative expression levels of 61.819-and 97.005-fold change in T3 and T4, respectively, revealed that anthocyanins played important roles in plant defense against viral infections. HPLC analysis showed that, a significant overaccumulation of the total phenolic contents in T4 (7,218.86 mg/kg) rather than T2 (1606.49 mg/kg) plants. Meanwhile, D' Amelia et al. 1 showed that AN2 able to regulate the production of phenolic compounds and high expression in potato tuber during drought stress was associated with increases in total phenolic levels 27 . The accumulation of anthocyanin in plants upon biotic stresses has been reported. Ustilago maydis triggered anthocyanin induction in maize 80 , and anthocyanin-enriched tomato fruits exhibited lower susceptibility to gray mold 81 . Moreover, antibacterial, antifungal and antiviral activities of certain anthocyanins were also reported [82][83][84] .
Bacillus species produce wide structural variability of secondary metabolites that exhibiting strong antibacterial and antifungal activities 85,86 . Moreover, it represents a new and rich source of secondary metabolites that need to be discovered. The GC-MS spectrum analysis showed that pyrrolo[1,2-a] pyrazine-1,4-dione was the major compound in POT1 ethyl acetate extract. Pyrrole was known for a wide range of bioactivities, including antibiotics, antitumor, antifungal, anti-inflammatory, anti-angiogenesis and cholesterol reducing drugs 87 . Moreover, pyrrolo[1,2-a]pyrazine-1,4-dione isolated from Streptomyces spp. showed antioxidant 88 and anticandidal 89 activity, while that isolated from Shewanella spp. exhibited anticyanobacterial and algicidal activity 90 . Additionally, these compounds showed excellent protease inhibitor activity with a very good antiretroviral activity 91 and have the ability to inhibit HIV-1 viruses, DNA polymerases and protein kinase activity 92,93 . The obtained results supported previous reports of pyrrolo [1,2-a] pyrazine-1,4-dione activity in preventing viral replication. Consequently, POT1 could be useful as a preventive agent against AMV infection. However, further examinations needed for the potential field application.  www.nature.com/scientificreports/ Source of bacterial isolate, biochemical tests characterization and culture filtrate preparation. Soil-adhered potato roots were collected from a potato field in Alexandria governorate, Egypt. The roots were crushed in a mortar and a loopful was cultured on Nutrient Agar (NA) media and incubated at 30 °C. Different colonies were picked and assayed for antiviral activity using half-leaf method 94 . The bacterial isolate showing a maximum antiviral activity was selected, and preliminary identified based on morphological and biochemical characteristics 95 . For the bacterial culture filtrate (CF), the selected bacterial isolate was grown in a nutrient broth medium and incubated on a shaking incubator at 30 °C for two days. The bacterial culture was centrifuged at 6,000 rpm for 10 min at 4 °C to separating bacterial cells and collecting supernatants.

Material and methods
DNA extraction, 16 rRNA amplification and sequencing analysis. Bacterial genomic DNA was isolated from bacterial culture of selected bacterial isolate using Wizard Genomic DNA Purification Kit (Promega, USA) according to the manufacture instructions. By using 16 rRNA specific primers, forward (5`-AGA GTT TGA TCC TGG CTC AG-3`) and reverse (5`-GGT TAC CTT GTT ACG ACT T-3`), PCR reaction was performed as previously reported 96 . Briefly, PCR reaction was started with initial denaturation at 95 °C for 2 min, followed by 35 cycles at 95 °C for 30 s, 50 °C for 30 s and 72 °C for 1.5 min. An additional final extension step was carried out at 72 °C for 5 min. PCR amplified products were checked on 1.5% agarose gel electrophoresis, visualized under UV transilluminator, and purified by a PCR clean-up column kit (QIAGEN, Germany) for sequencing. Sanger sequencing of 16 rRNA gene was performed using a BigDye Terminator v3.1 Cycle Sequencing kit and a 3130xl Genetic Analyzer system (Applied Biosystems, USA). After the sequencing process, the annotated nucleotide sequence was analyzed using NCBI-BLAST (https ://blast .ncbi.nlm.nih.gov/Blast .cgi), and deposited in Genbank. The phylogenetic tree was constructed based on the UPGMA statistical method with a bootstrap of 2.000 replicates using the MEGA 5 software 97 . replicates of each treatment were collected at 21 dpi and kept at -80 °C until use. Total RNA was extracted using the RNeasy plant mini kit (QIAGEN, Germany) according to the manufacturer's instructions. Each biological sample was a mix of five samples derived from five different plants. The extracted RNA was dissolved in DEPC-treated water, treated with RNase-free DNase to eliminate genomic DNA, quantified by NanoDrop UV spectrophotometer (Labtech International Ltd, Sussex, UK) and the integrity was assessed by agarose gel electrophoresis. Two micrograms of total RNA for each sample were reverse transcribed to cDNA using oligo (dT) and random hexamer primers with reverse transcriptase enzyme of Super-Script II (Invitrogen, USA), according to the manufacturer's instructions. The reverse transcriptase reactions were performed in a thermal cycler (Eppendorf, Germany), according to Behiry et al. 99 . The amplified cDNA was used as a template for quantitative real-time PCR (qRT-PCR).

Greenhouse experimental design.
Quantitative Real-time pcR (qRt-pcR) assay and data analysis. Different primer sets specific for polyphenolic-related genes were synthesized according to previous studies ( Table 5). The housekeeping gene EF1-α (Table 5) was used as a reference gene in order to normalization of the transcript expression levels 52 . Each sample in all reactions was run in triplicate using Rotor-Gene 6000 (QIAGEN, ABI System, USA) with the SYBR Green PCR Master Mix (Fermentas, USA) and performed according to previously reported 100 . The amplification program of the thermal cycler included an initial denaturation step at 95 °C for 10 min, followed by 40 cycles consisting of; denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s. After that, the melting curves were obtained to eliminate the inclusion of non-specific products. The relative expression level of the target gene was accurately quantified and calculated according to 2 -ΔΔCT algorithm 101 .
Accumulation level of AMV-CP. By using a specific primer of AMV-CP (Table 5), qRT-PCR was performed using the SYBR Green PCR Master Mix (Fermentas, USA) to detect AMV and its level in the tested potato leaves. The reaction consisted of a 20 μL mixture containing 1 μL cDNA (50 ng), 1 μL of 10 pmol μL −1 of each primer (forward and reverse), 10 μL of 2 × SYBR Green PCR Master Mix and 7 μL of nuclease-free water. The qRT-PCR reaction was performed using a Rotor-Gene 6000 in two steps. The first step at 95 °C for 10 min as initial denaturation step and the second step cycle consisting of 40 cycles (95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s). Each biological sample was run in triplicate to guarantee data reproducibility. The relative accumulation level of AMV-CP was calculated as according to described above.

GC-MS fractionation of bacterial ethyl acetate extract.
To identify active components of bacterial culture filtrate, a 48 h bacterial culture broth was precipitated, and the supernatant was collected and mixed with ethyl acetate, as a solvent, in the ratio of 1:1 (v/v). The mixture was shaken vigorously for 20 min, and by using separating funnel, the ethyl acetate phase was separated from the aqueous phase. Ethyl acetate extract was concentrated by evaporation at 50 °C in a rotary evaporator. The residue which contained the secondary metabolites and chemical compounds was analysed using gas chromatography-mass spectroscopy (GC-MS) 103 . The analyses were run on a GC-MS system (TRACE 1300 Series, Thermo, USA) and the test carried out at the Marine Pollution Lab of National Institute of Oceanography and Fisheries, Alexandria, Egypt. The mass detector used in split www.nature.com/scientificreports/ mode and helium gas with a flow rate of 1 ml/min was used as a carrier. The injector was operated at 250 °C and oven temperature for initial setup was at 60 °C for 2 min, scan time 0.2 s; mass range 50-650 amu and ramp 4/ min to 250 °C for 20 min. Mass spectra were taken at 70 eV, during the running time 53 min. The constituents were identified after comparing them with available data in the GC-MS library in the literature.

Statistical analysis.
The relative expression values of three replicates for each set were analysed by one-way ANOVA using the CoStat software. The significant differences of the relative expression levels were determined according to the least significant differences (LSD) P ≤ 0.05 level of probability, and standard deviation (± SD) is shown as a column bar. Compared to mocked-inoculated potato tissues, the relative expression values higher than 1 was demonstrated an increase in the gene accumulation (up-regulation), while values lower than 1 means a decrease in expression levels (down-regulation). www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.