Effect of Cytokinins on Micropropagation of Moringa Oleifera Lam. and Genomic Stability Assessment Using Flow-Cytometry

Moringa oleifera Lam. is a multipurpose medicinal plant of the family Moringaceae which has been widely utilized as a pharmaceutical remedy to treat a wide range of diseases. In addition, the tree has several applications in human nutrition as well as livestock feeding. M. oleifera is easily multiplied through epigeal germination (recalcitrant) but seed propagated plants are heterogeneous and take longer to reach fruit-bearing age. As an alternative, branch cuttings have been used but their establishment is erratic and often leads to reduced growth of the mother plant. Thus, to produce superior planting materials, in-vitro propagation has become paramount. As a result, several studies using a limited range of cytokinin have been undertaken to multiply M. oleifera through tissue culture. Otherwise, a study was conducted to examine the effect of five different cytokinins on in-vitro regeneration of this tree species. Results showed that nodal explants cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 6-Benzylaminopurine (BAP) and subsequently rooted on basic MS media was the most optimal treatment. Furthermore, acclimatization of plantlets in sterile soil substrate and perlite (1:3;v/v) under transparent polythene sheet for 7 days resulted in survival rate of 100%. Assessment of genetic fidelity using flow cytometry revealed that surface sterilization alongside cytokinin treatments produced plantlets that were genetically stable regardless of the growth regulator used. Thus, the in-vitro protocol developed in this study can be utilized for in-vitro studies and mass propagation of this imperative plant species.

Micropropagation of Feverfew (Tanacetum parthenium) and Quantification of Parthenolide Content in Its Micropropagated and Conventionally Grown Plants

Feverfew (Tanacetum parthenium) is a well-known multi-functional plant with anti-inflammatory, cardiotonic, antiangiogenic, and anticancer effects. The therapeutic value of this plant is due to its phytochemical constitutes, especially parthenolide. Tissue culture techniques have been applied to improve the bioactive components of many herbal plants. Hence, this study, was carried out to establish a protocol for micropropagation of the feverfew plant and to quantify parthenolide content in its micropropagated and conventionally grown plants. To establish an aseptic culture, different concentrations of sodium hypochlorite (NaOCl) were investigated for seed surface sterilization. Besides, the effects of plant growth regulators (PGRs) on the callus induction, shoot organogenesis from callus and in vitro rooting were evaluated. Additionally, the parthenolide yield of the micropropagated and conventionally grown plants was determined by using high-performance liquid chromatography (HPLC). The results showed that surface sterilization of feverfew seeds with 6% NaOCl for 15 min obtained 65.00 ± 2.69% aseptic seeds. Murashige and Skoog (MS) medium supplemented with 0.4 mg/L thidiazuron (TDZ) and 2 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) resulted in 86.00 ± 1.72% callus induction. The highest number of shoots (5.00 ± 0.15) per explant was obtained in the treatment of MS medium supplemented with 5 mg/L zeatin. MS medium fortified with 3 mg/L indole-3-butyric acid (IBA) produced the maximum number of roots per plantlet (8.90 ± 0.35). A total of 90% of the micropropagated plantlets survived when planted in perlite + peat moss (1:1 v/v); the micropropagated plantlets were successfully established in the ex vitro conditions. According to parthenolide analysis, its level was significantly higher in the micropropagated plants than conventionally grown plants. Among different solvents, ethanolic extraction obtained the highest parthenolide content of the feverfew plant. Hence, it can be concluded that micropropagation of feverfew could be applied to produce disease-free planting materials and to improve the parthenolide content of the feverfew plant.

GC-MS Profiling and Antifungal Activity of Secondary Metabolite from Endophytic Fungus of Giloy

The endophytic microbiota is considered to be one of the consistent and noble souce of potential and unique natural amalgams. These natural amalgams carry diverse pharmaceutical significance which the reason for their importance among research fields. The diversity of plants carries much more diversity of the endophytes as their mutual parts where both are benefited from each other. The current work deals with the isolation of the endophytic fungus from Tinospora cordifolia, for which the leaves were used after the surface sterilization, followed by the production of secondary metabolite by the endophytic isolates through submerged fermentation technique. The produced metabolite was extracted by liquid-liquid extraction technique, which was further used for evaluating its antifungal potential against Candida albicans and the obtained results show their considerable potential. The GC-MS profiling of secondary metabolite was conducted to determine the presence of some bioactive compounds in them, and as a result, some potential compounds detected are Levoglucosenone, Silanediol, Nonane, D-Allose, 5-Hydroxymethylfurfural. Since these compounds are biologically important in various aspects which gives the diversified application to the secondary metabolites. The study concludes the potential of secondary metabolites from endophytic fungus of Tinospora cordifolia and further investigation can be approached on determining the same from other plants, and also evaluating another bioactive potential of secondary metabolites.

Boeremia exigua Causes Leaf Spot of Walnut Trees (Juglans regia) in China

Walnuts are an important perennial nut crop widely cultivated in China, which are rich in protein, carbohydrate, renieratene, and other beneficial nutrients. China is the largest producer of walnuts in the world, with the largest planting area and output. At the end of April 2020, several unknown necrotic spots on leaves of walnut trees were observed in a Juglans regia field located in Sancha Town, Enshi, China (30°28′N, 109°64′E). Initially, lesions were black, small, sunken, and turning to yellowish-brown, irregular, well surrounded by brown margins. Severely, leaf spots coalesced and resulted in withered and abscised. In order to identify the pathogen, infected leaves were collected. Sections of leaves were aseptically excised from the margins of necrotic spots following surface sterilization and placed on potato dextrose agar (PDA) at 28℃. After 4 days, fungal isolates were obtained and purified by hyphal tip isolation. The isolates looked morphologically similar, producing colonies that appeared hyphae with dark grey, lobed margins, and aerial mycelium with white to light gray. After 15 days of incubation, subglobose, dark brown pycnidia (100-176 μm in wide, 75-95 μm in length) were formed with an orifice in the center, producing conidia. Conidia (3.5 to 9.0 × 1.6 to 4.5 μm) were oval to round, aseptate, occasionally 1-septate. These morphological characteristics lead to the conclusion that the isolates may be identified as Phoma sp. (Boerema et al. 1976). A single isolate was randomly selected and designated for further verification. To confirm the identity, the internal transcribed spacer region (ITS), actin (ACT) and beta-tubulin genes were amplified and sequenced ITS1/ITS4, ACT-512F/ACT-783R, and Bt2a/Bt2b, respectively (White et al. 1990, Groenewald et al. 2013). BLAST analysis of the ITS 505-bp sequence (GenBank accession no. MW282913), actin 269-bp sequence (GenBank accession no. MW201958), and beta-tubulin 347-bp sequence (GenBank accession no. MW273782) showed ≥99% homology with the sequences of B. exigua available in GenBank (GenBank accession no. AB454232, LT158234, and KR010463, respectively). Base on the above results, the strain HTY2 was identified as B. exigua. Pathogenicity was tested. Walnut plants were spray-inoculated with a spore suspension (5 x 105 CFU/mL). Controls were inoculated as described above except that sterile distilled water in the dark at 25 ℃. After seven days, lesions were evident at inoculation points, and equivalent to those observed in field were observed. Control leaves remained symptomless. The pathogenicity test was repeated thrice and the results were the same, fulfilling the Koch’s postulates. The pathogen has been reported on various plants around the world, causing a series of symptoms. Infected plants rarely died, but the presence of lesions decreased their fruit quality and yield. Previous identification of the disease is essential in formulating management strategies.

Flower endophytic fungi of Geodorum densiflorum endangered orchid

Geodorum densiflorum is an ornamental and medicinally important orchid. The medicinal metabolites are produced by endophytic fungi associated with orchid tissue, particularly flowers. However, there is no report of the endophytic fungi from the G. densiflorum flowers. This research aimed to study the endophytic fungi from different parts of G. densiflorum flower. Fungal isolation was carried out from sepal, petal, labium, stigma, and anther using surface sterilization method. The fungi were identified by combining morphological and molecular characteristics of ITS rDNA region. The results showed that each flower organ had different species of endophytic fungi. A total of seven species of endophytic fungi were obtained; four species were successfully identified by molecular identification and three species based on morphology. The four species, namely Hypomontagnella barbarensis, Aspergillus oryzae, Curvularia pseudobranchyspora, and Nigrospora chinensis, while the three species, namely Gonatobotrys sp., Humicola sp., Aspergillus section Nigri. The labium inhabited by Curvularia pseudobranchyspora, Nigrospora chinensis, Aspergillus section Nigri. Three species isolated from petals, namely Hypomontagnella barbarensis, Gonatobotrys sp., and Aspergillus oryzae. The sepal is inhabited by Humicola sp. There were no endophytic fungi in stigma and anther. This indicates that each flower part is a unique habitat of endophytic fungi.

Secondary Metabolite and Antioxidant Activity of Endophytic Fungi Isolated from Syzygium aqueum Leaves Stalk

Medicinal plants are a promising host for endophytic fungi to produce secondary metabolites relevant for food and health. In this study, we evaluate antioxidant activity to determine the species of endophytic fungi isolated from Syzygium aqueum. Endophytic fungi were isolated from leaf stalks through surface sterilization. The fungi’s isolate was identified with morphology and molecular analysis (ITS-rDNA). The pure fungi strain was cultivated on PDB media for 4 weeks, and metabolites were extracted using ethyl acetate. The crude extract of endophytic fungi was examined for its antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH). The pure compound was isolated using the chromatography method, and its structure was determined using spectroscopy analysis involving NMR 1D and 2D. In total, four obtained endophytic fungi were isolated from leaf stalks. The fungi with good antioxidant activity (IC50 59.2 µg/mL) were identified as Beltrania rhombica. The characteristics of the pure compound are white-yellowish powder with IC50 44.2 µg/mL. Based on spectroscopy analysis, the pure compound was identified as 3-(hydroxyl(3,4,5-trihydroxylphenyl)methyl)-3,4-dihydro-2H-pyran-4,5,6-triol.

Optimization of cultural conditions affecting improved bioactive metabolite production by endophytic fungus Trichoderma harzianum

The present study aimed to optimize cultural conditions for optimum bioactive metabolite production by endophytic fungus Trichoderma harzianum, isolated by surface sterilization method from the leaf of the eucalyptus plant. The fungus was identified based on morphological characterization. Fungal metabolites were carried out by ethyl acetate solvent. The antibacterial activity was tested against Escherichia coli (ATCC 25922) and Staphylococcus aureus (NCTC 6571). Various carbon, nitrogen sources, pH, temperature, incubation period, and NaCl on the antibacterial metabolite production were studied. Bioactive metabolite production of T. harzianum exhibits a broad spectrum of in vitro antibacterial activity against two strains of bacteria. For the optimum production of bioactive metabolites, Dextrose and Glucose were found to be the best sources of carbon and the best sources of Nitrogen Yeast extract (YE) and (NH4)2SO. The maximum production of bioactive metabolites occurs at pH 7 and 25°C.; the NaCl showed a positive influence on bioactive metabolites.

Determination of Salmonella enterica Leaf Internalization Varies Substantially According to the Method and Conditions Used to Assess Bacterial Localization

In a previous study, comparing the internalization of S. enterica serovar Typhimurium in various leaves by confocal microscopy, we have demonstrated that the pathogen failed to internalize tomato leaves. Numerous reasons may account for these findings, yet one such factor might be the methodology employed to quantify leaf internalization. To this end, we have systematically studied leaf localization of a Green-fluorescent protein-labeled Salmonella strain in tomato, lettuce, and Arabidopsis leaves by surface sterilization and enumeration of the surviving bacteria, side by side, with confocal microscopy observations. Leaf sterilization was performed using either sodium hypochlorite, silver nitrate, or ethanol for 1 to 7min. The level of internalization varied according to the type of disinfectant used for surface sterilization and the treatment time. Treatment of tomato leaves with 70% ethanol for up to 7min suggested possible internalization of Salmonella, while confocal microscopy showed no internalization. In the case of in lettuce and Arabidopsis leaves, both the plate-count technique and confocal microscopy demonstrated considerable Salmonella internalization thought different sterilization conditions resulted in variations in the internalization levels. Our findings highlighted the dependency of the internalization results on the specific disinfection protocol used to determine bacterial localization. The results underscore the importance of confocal microscopy in validating a particular surface sterilization protocol whenever a new pair of bacterial strain and plant cultivar is studied.