1. Introduction
Cancer is one of the greatest widespread destructive diseases, influencing millions of people each year, and it has been anticipated as the second principal causative agent of human death following cardiovascular disease [
1]. In 2018, about 18.1 million people worldwide had cancer, and 9.6 million died from the disease, those numbers are projected to nearly double by 2040 [
1]. Global annual expenses on anticancer drugs are approximately
$100 billion and are predicted to rise to
$150 billion by 2020 [
2].
Periwinkle (
Catharanthus roseus L. (G.) Don; Apocynaceae), is a significant source of high natural antioxidants and terpenoid indole alkaloids (approximately 130 TIAs) involving two essential dimeric alkaloids utilized in cancer chemotherapy [
3]. The most important periwinkle alkaloids are vincristine (VCR) and vinblastine (VLB) which have been applied as a foremost active constituent of different marketable chemotherapy drugs (ONCOVIN and VELNR respectively) for chronic cancers such as leukemia, breast carcinoma, lung cancer, and Hodgkin’s disease [
3,
4]. Formerly, they were isolated in trace amounts (0.0003% and 2.56% of dry leaf weight for VCR and VB respectively) from periwinkle leaves [
5]. Additionally, it contains numerous imperative bioactive constituents—such as anthocyanins, flavonol glycosides, phenolic acids, saponins, steroids, and terpenoids—that display antidiarrheal, antidiabetic, antihyperglycemic, antimicrobial, wound healing, and antioxidant activities [
6,
7]. Periwinkle plant water extracts are disbursed for several applications, i.e., diabetes, fever, or rheumatism [
8]. Additionally, the plant leaves have been ground to suppress feelings of hunger and fatigue [
9]. Phenolics represent the most plentiful and widespread plant natural products, occupying imperative purposes for the plant, i.e., defense against environmental stresses, herbivore restriction, and signaling in plant–microbe interactions. For humans, plant phenolics are a source of numerous plant-derived drugs and they have recently attracted more consideration owing to their implication in protection against cancer, cardiovascular, and neurodegenerative diseases, in relation to their antioxidant action [
10,
11].
To keep up with the growing requirements for anticancer drugs and the attendant challenge of their production costs (from
$1–3.5 million kg
−1), widespread interest over the past 25 years has intensified for increasing their production [
2]. Due to intricate structures of the alkaloid (particularly VLB and VCR), their chemical synthesis at a large scale is not efficiently practicable [
9]. Therefore, various approaches—i.e., in vitro cultures, metabolic engineering [
12,
13], and semi-chemical synthesis [
14]—are being explored to achieve improved specific indole alkaloid production. Semi-synthesis and derivatization of intricate biochemical construction by chemical methods are also reliant upon TIA precursors that are likewise fed-up from medicinal herbs [
14]. The reconstitution of herb metabolic pathways in heterogonous hosts has led to restricted achievement [
15]. Distinction of intracellular compartmentalization and enzymes involved in TIA biosynthetic pathways to the production of the end product are still unexplored through system biology approaches [
16]. One of the most imperative ways for improving the assimilation of secondary metabolites and phytopharmaceuticals is the utilization of elicitors [
17,
18]. Elicitors are biotic or abiotic materials that are able to induce an enhancement in the assimilation of secondary metabolites via the stimulation of defensive responses, biochemical modification, and accretion of phytoalexin. There are several abiotic elicitors (metal ions, inorganic compounds, and precursors) and biotic elicitors (endophytes microbes, plant cell wall components) that are normally applied [
5,
17].
Plant endophytes colonize and spend their entire life cycle or part of their life cycle within the healthy plant tissues devoid of any obvious infection symptoms or noticeable manifestation of diseases, also, they could be a promising resource of novel natural products for medicinal, agricultural, and industrial uses [
14,
18,
19]. The endophyte microbes enter the plant through root hairs or the leaf stomata followed by systemic distribution all over the plant. Endophytes can produce plant growth substances, accelerating nutrient availability and antioxidant capacity that induces plant growth and secondary metabolite accumulation [
20,
21,
22]; however, there is very little pre-existing research on endophyte utilization for improving the phytopharmaceutical biosynthesizing capacity in periwinkle plants.
Recently, feedings with specific precursors (i.e., tryptophan) have proved to be practical and successful approaches for raising the level of phytopharmaceuticals, including alkaloids [
23]. Tryptophan application motivates the growth and biochemical processes of numerous plants by accelerating indole acetic acid biosynthesis, as well as increasing the content of chlorophyll, soluble and insoluble sugars, as well as total alkaloids [
24,
25]. Chitosan has attracted massive deliberation as a significant biological resource owing to its biological features—i.e., non-toxicity, biodegradability, and eco-friendliness—with different usages in agriculture [
26,
27]. Recently, a few reports have revealed that chitosan has been established as an efficient abiotic elicitor for improved plant growth, activating antioxidant capacity, as well as enhancing secondary metabolites production in diverse plants [
26,
28]. Additionally, chitosan application increases the superoxide dismutase activity and reduces oxidative biomarkers [
28,
29,
30]. Inorganic chemicals, i.e., aluminum chloride, have been widely applied in several herb species for the production of phytopharmaceuticals via changing plant secondary metabolism processes [
13,
26]. Aluminum, a noxious soil metal, was formerly recognized as a biogenic elicitor that upregulates genes associated with plant defense strategies under environmental stresses [
31].
Previous studies have separately recognized that endophytes or abiotic elicitors, as a cost-effective agent, have multiple biochemical functions in plant development and biochemical pathways as well as secondary metabolite assimilation. Conversely, their integrative application in inducing phytopharmaceutical production in periwinkle plants to our knowledge has not been documented. Therefore, the current study aims to examine whether the application of abiotic elicitors with or without endophytes could be a valuable strategy for improving the biomass and phytopharmaceutical production of the periwinkle herb.
2. Materials and Methods
The current study was done in the research farm and lab of the Agricultural Botany Department, Mansoura University, Egypt (latitude 31°02′40.6″ N, longitude 31°22′40.3″ E, altitude 15 m above sea level), in the 2018 and 2019 seasons, to evaluate the role of two periwinkle endophyte microbes (Streptomyces sp. and Bacillus sp.) with or without abiotic elicitors (aluminum chloride, tryptophan, and chitosan) on the plant biomass, physio-biochemical attributes, phytopharmaceutical constituents, and alkaloid production.
2.1. Endophyte Micro-Organisms Isolation, Selection, and Identification
Healthy periwinkle plants were collected from the Mansoura University garden, to isolate the endophytic microbes. The plant shoots were washed thoroughly in sterilized distilled water (SDW) and then surface-sterilized for 1 min in 70% ethanol, 2.5% sodium hypochlorite, 70% ethanol respectively, and finally rinsed in SDW three times to ensure that all isolated microorganisms are endophytes and to kill saprophytic or parasitic microbes [
32]. To validate the surface sterilization efficiency, we used the protocols described by Coombs and Franco [
33].
The surface-sterilized shoot segments (1 cm long from stems and leaves) were divided into three parts, the first part was placed in Petri dishes (9.00 cm) with potato dextrose agar (PDA) with 0.003 mL/l rose bengal and streptomycin (250 mg/L) to isolate the endophytic fungi and then incubated at 26 ± 2 °C until growth was observed. The second part was crushed in SDW for isolation of the endophytic bacteria. An aliquot (1 mL) of sterilized crushed samples was spread onto nutrient agar media (NA) plates and incubated at 30 ± 2 °C [
34]. The third part was crushed as mentioned before, spread onto starch nutrient agar (SNA) plates at a dilution of 10
−6, and incubated at 28 ± 2 °C to isolate the endophytic actinomycetes. Regular observations for endophyte growth were done from the 2nd to 10st days from inoculation. Individual hyphal tips of the various fungi from internal tissues or colonies from crushed segments were removed and cultured again on PDA, NA, and SNA plates, and subsequently incubated at 28 ± 2 °C for at least 10 days. All endophyte cultures were assessed for purity, transferred to fresh culture slant by hyphal tips, as well as a single spore, and stored at 4 °C for additional observations [
35].
Identification of endophyte isolates was done depending on the morpho-taxonomical features and microscopic observations of the mycelia, spore and colony shapes, Gram staining, spore formation, and colony pigmentation via the regular mycological guides [
36,
37]. For tentative identification, microscopic slides were prepared and checked under a binocular compound microscope for morphological identification. Numerous online databases (MycoBank, Fungal Planet; Index Fungorum, Bibliography of Systemic Mycology) are useful in the identification of fungal isolates. On the other hand, the physio-biochemical features of bacterial isolates were determined following
Bergey’s Manual of Determinative Bacteriology (eighth edition). A total of 15-endophyte microorganisms were recognized. For choosing the most effective endophytic isolates, we prepared an initial experiment in the greenhouse. This experiment contained 16 groups (each one containing three pots) for 15 endophytes and a non-inoculated treatment.
For bacterial inocula preparation, isolated bacterial strains were grown on liquid NA to maximize bacterial cell numbers for 2 days at 30 ± 2 °C. Subsequently, the bacterial cultures were collected by centrifugation (3000 rpm for 30 min) at lab. temperature. The sediment was re-suspended in 50 mL of 0.8% potassium chloride solution, then the concentration of bacteria was adjusted to 10−8 cell mL−1 with SDW. Meanwhile, the isolated fungi and actinomycete strains were grown on a solid medium (PDA and SNA respectively) and incubated at 28 ± 2 °C for 5 days. To inoculum preparation, spores were collected from the surface of the medium and suspended in sterile potassium chloride (0.9%) solution. Spore suspension (100 μL from a 106 cfu/mL suspension) was transferred to a 50 mL liquid medium and incubated in a rotary shaker incubator at 150 rpm (at 28 ± 2 °C). After 3 days, the culture was centrifuged at 3000 rpm for 10 min. the sediment was re-suspended in a 50 mL sterile solution (containing 0.8% potassium chloride), then the concentration of spores ml−1 was adjusted to 1.5 × 106 with SDW. The inoculums were mixed well with the experimental soil at 20 mL kg−1 soil and the pots were filled. Prepared inoculums were mixed well with the experimental soil at 10 mL kg−1 soil and the pots were filled separately. Then the healthy and uniform periwinkle seedlings (5/pot) were transplanted. Ten mL of inoculums were then added to each pot after 10 days following transplanting. At the end of the experiment (30 days following transplanting), the plants were collected for estimation of shoot dry weight and alkaloid content. Depending on the obtained results from this experiment, two endophyte isolates were chosen for the main investigation as indicated below.
The two selected isolates were identified by 16S rRNA gene sequences. The genomic DNA of endophytes was extracted [
38], and 16S rDNA was amplified in polymerase chain reaction using the genomic DNA as a template and universal primers, 27F (5′GAGTTTGATCACTGGCTCAG-3′) and 1492R (5′TACGGCTACCTTGTTACGACTT-3′) [
39]. To know the identity of isolates, obtained sequences were compared with nucleotides via GenBank [
40]. The selected endophytes were
Bacillus sp.-JN256920 and
Streptomyces sp.–HE591384.
2.2. Plant Material and Experimental Layout
Outdoor pot experiments were done at the experimental farm of the Agricultural Botany Department, Mansoura University, Egypt, in a completely randomized block design. The experiment is composed of two factors; the first factor includes endophyte micro-organisms (no endophytes, Bacillus sp., Streptomyces sp.). The second factor involved the abiotic elicitors (no-elicitors ‘water’, aluminum chloride, tryptophan, chitosan). Therefore, the experiment consisted of 12 treatments replicated five times. The appropriate abiotic elicitor concentration in the present research was based on previous studies.
The plastic pots (30 cm inner diameter) were filled with 7 kg clay loam soil (43.54% clay, 34.69% silt; 21.77% sand). The physicochemical properties of the experimental soil were pH (1:2.5 soil suspension,
w/
v) 7.43 and 7.46; nitrogen (N) 208 and 209 mg kg
−1; phosphorus (P) 5.3 and 5.4 mg kg
−1; potassium (K) 179 and 181 mg kg
−1; organic matter 1.03% and 1.05%, in the experimental seasons respectively, based on the protocols summarized in Motsara and Roy [
41]. Each pot was supplemented with a basal amount of NPK fertilizer (26-13-22 mg kg
−1 soil) as ammonium sulfate, single superphosphate, and K-sulfate at the time of planting, which was repeated each month.
Seeds of periwinkle plants were sourced from the Horticulture Research Institute, Egypt. Thirty sterilized seeds were sown in each pot on 15 February each year and then irrigated regularly (90–100% of field capacity). After one month, the seedlings in each pot were thinned to leave 10 healthy and uniform seedlings, and subsequently thinned again to 3 plants pot
−1 45 days after sowing. At 60 days after sowing, the pots were divided into 12 groups, each one including 5 replicates as indicated in
Figure 1 for endophytes and abiotic elicitors’ treatments. The
Bacillus sp. (CFU of 10
−8 mL
−1) and
Streptomyces sp. (CFU of 1.5 × 10
6 mL
−1) inoculum (as mentioned previously) were poured into the soil in irrigation water (30 mL pot
−1), then mixed with the upper soil surface. Foliar spraying of different elicitors (133 mg L
−1 aluminum chloride; 150 mg L
−1 tryptophan; and 500 mg L
−1 chitosan) with Tween 20 as a wetting agent was done three times in 20 days intervals (60, 80, and 100 days from sowing) until dripping using a hand sprayer. Five plants for every treatment were harvested at 120 days from sowing for plant growth, physiological attribute, and secondary metabolite assessment.
2.3. Growth Parameter
The plant samples were collected carefully, cleaned with SDW, and then shoot fresh (FW) and dry (DW) weights were determined.
2.4. Total Chlorophyll and Carotenoid (mg g−1 FW)
The photosynthetic pigment was extracted and estimated following Lichtenthaler and Welburn [
42] protocol. Commonly, 200 mg FW from the fourth upper leaves on the main stem were rinsed for 24 h in pre-cooled methanol (96%) supplemented with sodium bicarbonate (0.05%). The absorbance was recorded at 470, 653, and 666 nm spectrophotometrically (T60 UV–Visible spectrophotometer, UK).
2.5. Shoot Ion Percentage
Nitrogen (N), phosphorus (P), and potassium (K) were extracted from the plant shoot and then estimated following the protocol of Motsara and Roy [
41]. About 200 mg of shoot dry powder was carefully transferred to a digestion flask containing 5 mL of concentrated H
2SO
4. Digestion was done at 100 °C for 2 h, subsequently; the mixtures were chilled for 15 min at lab. temperature. Then, an aliquot of H
2SO
4/HClO
3 mixture was added dropwise. Total N and K were determined with the micro-Kjeldahl method and flame-photometrically respectively. The scheme of Cooper [
43] was followed to determine the P in the digested samples against the phosphate standard curve.
2.6. Oxidative Biomarkers
Hydrogen peroxide (H
2O
2; μM g
−1 FW) in the shoot was determined spectrophotometrically as described by Tariq et al. [
44] using titanium reagent. The absorbance was recorded at 415 nm against a blank. The hydrogen peroxide concentration was calculated according to a standard curve of H
2O
2.
Lipid peroxidation (µM malondialdehyde g
−1 FW) was estimated following the Djanaguiraman et al. [
45] method using a thiobarbituric acid reagent. The malondialdehyde concentration was deliberate via an extinction coefficient of 155 mM
−1cm
−1.
2.7. Antioxidant and Phytopharmaceutical Constitution
For estimation of ascorbic acid (mg g
−1 FW), homogenization of shoot fresh tissues was performed in oxalic acid and thereafter centrifuged, the supernatant was stored for the assessment of ascorbic acid. A Sadasivam and Manickam [
46] method was used to determine the concentration of ascorbic acid in a periwinkle shoot with 2, 6- dichlorophenol indophenol reagent following the equation
where:
a, weight of sample;
b, volume made with metaphosphoric acid;
c, volume of aliquot taken for estimation;
d, dye factor;
e, average burette reading for sample
Antioxidant enzymes and soluble protein were extracted from plant tissues with K-phosphate buffer (100 mM, pH 7.0) following the methods of Chrysargyris et al. [
47].
Catalase activity (EC 1.11.1.6; unit mg
−1 protein) was assayed in a 3 mL reaction volume containing 1 mL of 50 mM K-phosphate buffer (pH 7.5), 0.1 mL EDTA, 0.2 mL enzyme extract, and 0.1 mL hydrogen peroxide. The activity was estimated spectrophotometrically at 240 nm [
48], 1 unit catalase =1 mM of H
2O
2 decline min
−1. The activity of peroxidase activity (EC 1.11.1.7) was estimated spectrophotometrically at 436 nm with guaiacol as a substrate in the existence of hydrogen peroxide using the method of Zhang et al. [
49]. The peroxidase activity (unit mg
−1 protein) was determined using the coefficient of extinction of 2.47 mM cm
−1 (1 unit =1 μmol of H
2O
2 decay min
−1). Soluble protein was estimated in the extract following the technique of Bradford [
50].
Total soluble phenolic compounds (mg gallic g
−1 DW) were assessed following the procedure described in Sadasivam and Manickam [
46]. Plant samples (1.0 g DW) were extracted with 10 mL ethanol (80%) with a pre-cooled pestle and mortar. The mixture was centrifuged and then evaporated to dryness. Afterward, Folin–Ciocalteu reagent (0.5 mL) and Na
2CO
3 (20%) were added to every tube. The absorbance was recorded at 650 nm aligned with a blank.
Total flavonoid (mg quercetin g
−1 DW) was estimated with AlCl
3 technique [
51]. To 5 mL plant extracts, 0.3 mL of sodium nitrite (5%,
w/
v) was added and 3 mL of AlCl
3 (10%). Subsequently, 2 mL of sodium hydroxide (1 M) was added and mixed well, and then the absorbance at 415 nm was recorded by spectrophotometer.
The total anthocyanin (mg 100 g
−1 FW) was estimated by Abdel-Aal and Hucl [
52] method. One gram of fresh herb tissues was homogenized with 5 mL pre-chilled acidified methanol (1% HCl), then centrifuged. The supernatant absorbance was estimated at 530 nm via spectrophotometrically. The total anthocyanin concentration was calculated as cyaniding-3-glucoside based on Abdel-Aal and Hucl [
52] equation.
For total alkaloid determination, a dry powdered shoot was extracted with acetic acid (10%) in ethanol for 4 h, then filtered and concentrated to one-quarter of the original quantity in a water bath. Ammonium hydroxide (AH) was added drop-wise for the entire precipitation and the solution was permitted to stand. The collected precipitates were washed with dilute AH and then filtered, dried, and weighed [
53]. Total alkaloid percentage (TAC) was determined by the equation
After that, the alkaloid yield (mg plant
−1) was calculated by the formula
2.8. Statistical Analysis
Homogeneity of error variance for all variables was done before the analysis of variance (ANOVA). The outputs displayed that all data fulfilled the homogeneity to achieve additional ANOVA tests. The data were statistically analyzed by a two-way ANOVA, at a 95% confidence level, using CoHort Software, 2008 statistical package (CoHort software, 2006; release, Cary, NC, USA). The statistical significance was considered as: * p ≤ 0.05, ** p ≤ 0.01; *** p ≤ 0.001, and ns (not significant). The difference between treatment means was assessed by Tukey’s HSD Multiple Range Test at p ≤ 0.05. The values are presented in tables as the means ± standard error (SE).