Allium schoenoprasum, which is commonly known as chive, is a perennial plant that is used as a popular seasoning and is cultivated globally . Phytochemical analyses of A. schoenoprasum extracts revealed that its water extract contains flflavonoid compounds, glycosides, and saponins while the methanol and ethyl acetate extracts contain alkaloids, flavonoids, glycosides, and saponins . Chive leaves have great medicinal values, such as positive effects on the circulatory system by reducing blood pressure and antimicrobial effects . Chive leaf infusions, often rich in potassium, are used for treatment of kidney stone disease by dissolving calcium oxalate in kidneys . Kucekova et al. demonstrated that chive flower extract obtained by solid-liquid extraction have a great effect on human keratinocyte cell line by decreasing cell proliferation, perhaps, due to their phenolic compounds. It is worth-mentioning that chives may contain many of the bio-active phenolic compounds such as gallic acid, coumaric acid, ferulic acid, and rutin that are commonly found in other Allium spp. Hence, the objective of this study was to assess plant growth of chive and secondary metabolites of chive leaves and roots following cultivation in a plant growth medium inoculated with conidia of an endophytic entomopathogenic fungus under greenhouse conditions. This is the first study on the experimental inoculation of chive with an endophytic fungus, vertical hydroponic nft system and its subsequent effects on plant growth and production of secondary metabolites.An indigenous B. bassiana strain that was previously isolated from a soil sample collected from the Cape Winelands in South Africa was used in this study. This strain was identified using molecular and morphological techniques as described in Moloinyane and Nchu .
The strain is being maintained at Cape Peninsula University of Technology in Bellville, South Africa. The fungus was cultivated on half-strength potato dextrose agar ; 0.02 g/L of ampicillin , and 0.04 g/L streptomycin in 9 cm and 14 cm diameters petri dishes. Fungal cultures were incubated at 25 C in the dark for four weeks. Using a spatula, mature four-week-old B. bassiana conidia obtained from PDA plates were transferred to 2 L glass bottles containing sterile 0.01% Tween 80 and sterile water. Bottles were capped, mixed by shaking for 5 min and by using a magnetic stirrer to homogenous conidial suspensions. The conidia inoculum concentration was enumerated using a haemocytometer and observed with a light microscope at 400X magnification. In order to obtain the desired concentration , the volume of sterile 0.01% Tween was increased or conidia were added to the glass bottle. This was followed by 10-fold serial dilutions to obtain lower concentrations of conidial inoculum suspensions; 1 104 , 1 103 conidia mL 1 . A conidial germination test to determine conidial viability was carried out according to the method described by Inglis et al. and high spore germination of over 90% was obtained.Chive seedlings, were purchased from Stodels Nurseries Ltd in Bellville, Western Cape Province, South Africa. Plants were maintained in the greenhouse at Cape Peninsula University of Technology in Bellville, South Africa at 23–25 C, 60%–80% RH and 13/11 natural light/dark regime. Individual chive plants were separated from a clump and one plant was transplanted to each 10 cm diameter pot containing a substrate mixture of one-third of river sand, one-third of vermiculite, and one-third of perlite by volume. The substrate materials were sterilized using 1% sodium hypochlorite for 1h before rinsing with sterile distilled water. The plants were fed with water soluble, formulated hydroponic fertilizer, Nutrifeed . The fertilizer was dissolved in sterile distilled water at a concentration of 10 g/5 L and 100 mL of the mixture was added to each plant once a week. Each plant was watered with 100 mL reverse osmosis water once a week.Leaf length from the soil surface to the top of the highest leaf and leaf number of each plant were measured weekly for three weeks.
Leaf length increment was calculated as the difference between leaf length at weeks one and three and percentage growth was calculated as follows: 100 x the leaf length increment divided by week one leaf length. Similarly, leaf number was counted and leaf number increment and % increase were calculated. At the end of the experiment, three weeks post fungal inoculation, root length and fresh weights of plants were recorded.Dry weights of the plants were determined by placing plants in paper bags in a drying oven at 35 C for 7 days.To determine fungal colonization of leaf tissue, three sections of leaf as well as root sections were collected from each plant soon after harvesting. The excised material was surface sterilized by first dipping in 70% ethanol for 3 s and then rinsing in sterile distilled water for 1 min. Thereafter, the sterile leaf and root sections were placed on solid half-strength PDA, incubated in the dark at 25 C, and were checked for outgrowth of B. bassiana in root and leaf tissues under stereo microscope after one and two weeks. efficiency of surface sterilization was evaluated by placing drops of the previously used 70% ethanol and distilled water for surface sterilization on plates containing solid half-strength PDA, and then incubated and checked for fungal outgrowth.The spectroscopic method described by Fadhil and Reza was used to determine total alkaloids in the plant extracts. Briefly, 100 mg of the chive leaf and root materials were extracted separately with 10 mL of aqueous ethanol for 2 h, centrifuged and the supernatant was used in the assay. Two milliliters of the extract supernatant and atropine standard solutions were mixed with 5 mL sodium phosphate buffer and 12 mL bromocresol green solution. Thereafter, 12 mL of chloroform was added to the solution and the solution was mixed vigorously using a vortex mixer. The absorbance at 417 nm was determined and the concentration of mg atropine equivalent per g dry weight in the sample using a standard curve of atropine was calculated. The total polyphenol content of the aqueous ethanol extracts of dried leaf and root dried materials of the chives were determined by the FolinCiocalteu method . The method of Swain and Hills was adapted for the plate reader.
Using a 96-well microplate, 25 μL of the sample was mixed with 125 μL Folin-Ciocalteu reagent and diluted 1:10 with distilled water. After 5 min, 100 μL aqueous Na2CO3 was added to the well. The plates were incubated for 2 h at room temperature before the absorbance was read at 765 nm using a Multiskan plate reader . The standard curve was prepared using 0, 20, 50, 100, 250 and 500 mg/L gallic acid in 10% ethanol and the results were expressed as mg gallic acid equivalents per g dry weight . The flavonol content of the aqueous ethanol extracts of dried leaf and root materials of the chives were determined using quercetin 0, 5, 10, 20, 40, and 80 mg/L in 95% ethanol as standard. In the sample wells, 12.5 μL of the crude aqueous extracts were mixed with 12.5 μL 0.1% HCl in 95% ethanol, and 225 μL 2% HCl and incubated for 30 min at room temperature. The absorbance was read at 360 nm, at a temperature of 25 C . The results were expressed as mg quercetin equivalent per g dry weight .The B. bassiana isolate used in the present study was able to endophytically colonize chive plants. The re-isolation of the fungus from chive leaf samples showed that the fungus was systemic, i.e., inoculum was transferred from the growth medium to the leaves. This is the first record of successful experimental inoculation and colonization of B. bassiana in chives. Previous studies have reported the colonization of B. bassiana in other plant species with different inoculation methods . Inoculation with B. bassiana did not improve the growth of chives over the control treatment . In fact, the control plants had slightly higher biomass and root growth compared to the treated plants. These results differ from those reported in previous studies, which showed that B. bassiana promotes plant growth of cassava , faba bean and cotton . However, Lewis et al. reported no significant difference in the growth of maize plants exposed to seed treatments with B. bassiana and the corresponding control treatment. Jaber and Enkerli reported inconsistent endophyte-induced plant growth promotion across sampling dates following foliar inoculation of faba bean plants with B. bassiana , B. brongniartii and M. brunneum . The root lengths varied significantly among treatments, and the shortest length was observed in plants exposed to the highest concentration of fungal treatment, which may suggest that fungus might have had a negative effect on root length. In a more recent study, B. bassiana inoculation had a positive influence on plant growth parameters including root length of common beans .
Nevertheless, the colonization of plant tissues by fungal endophytes can be influenced by many factors, such as inoculation method, species and fungal strain . Fungal endophytes might affect the nutrient cycle and uptake of nutrients from the soil by plants . In this study, chemical analysis revealed that the leaves and roots of both B. bassiana-exposed and unexposed chive contained polyphenols, nft hydroponic system alkaloids and flavonols, and also that these secondary metabolites were significantly more concentrated in the leaves than in the roots. Chives and other Allium species, such as onion and garlic contain polyphenols, alkaloids, flavonoids, glycosides and organosulfur . Furthermore, higher alkaloid content was detected in the leaves of plants inoculated with B. bassiana than in the control treated plants . In a previous study, which involved the same fungal strain used in this study, drenching potted grapevine plants with its conidial suspension induced higher production of anti-insect volatile compounds including Naphthalene in the fungus-exposed plants compared to the control . The better yield of alkaloids in fungus-treated plants could be due to the synthesis of secondary metabolites by fungus in the plant tissues . Also, endophytes can potentially induce host plants to accumulate secondary metabolites . Zhang et al. reviewed a wide range of bio-active alkaloids that are produced by endophytic fungi. Lozano-Tovar et al. reported that B. bassiana produce secondary metabolites that can induce anti-fungal activity. B. bassiana produces several biological active metabolites of the class of alkaloids such as tennelin, bassianin, pyridovericin, and pyridomacrolidin .
In the current study, since the specific alkaloid compounds were not detected, it is not possible to establish with certainty whether the higher total alkaloid content detected in the fungus-treated plants in this study was due to the direct production of alkaloids by B. bassiana or the fungus physiologically influenced the plant cells to produce more alkaloids. It is worth mentioning that fungal endophytes can produce mycotoxins in their host that are potentially harmful to livestock and humans . In conclusion, this study demonstrated that endophytic entomopathogenic fungi could be used to improve the yield of alkaloids in medicinal plants. In order to further understand the influence of fungal endophytes on plant production of bio-active compounds, future studies involving detailed phytochemical elucidation of the bio-active constituents of fungus-treated plants are warranted.The accumulation of biosynthesised secondary metabolites in plants could enhance the value of plant-based medicinal materials . However, the physiological and morphological processes in plants are affected by ambient environmental factors. Water, for example, is an essential resource for plant growth and survival, and its availability influences plant physiological processes including biosynthesis of secondary metabolites and enzyme activities.Water has many essential roles in plants: it is used for translocation and distribution of nutrients and metabolites, it maintains rigidity of plant organs, it is a medium for chemical reactions, and it is an essential component of the photosynthetic process . Water deficit is the most important limiting factor of plant growth .Water deficits can affect photosynthesis through stomata closure and decreased CO2 diffusion to the chloroplast . Drought stress is also associated with the enhancement of accumulation of many classes of natural products in plants, such as terpenes, phenols, alkaloids and glucosinolates.Leaves of water-stressed Ctenanthe setosa tended to accumulate more carbohydrates of low molecular weight and phenolic acids .