Zinc (Zn) is one of the most abundantly found heavy metals in the Earth’s crust and is reported to be an essential trace metal required for the growth of living beings, with it being a cofactor of major proteins, and mediating the regulation of several immunomodulatory functions. However, its essentiality also runs parallel to its toxicity, which is induced through various anthropogenic sources, constant exposure to polluted sites, and other natural phenomena. The bioavailability of Zn is attributable to various vegetables, beef, and dairy products, which are a good source of Zn for safe consumption by humans. However, conditions of Zn toxicity can also occur through the overdosage of Zn supplements, which is increasing at an alarming rate attributing to lack of awareness. Though Zn toxicity in humans is a treatable and non-life-threatening condition, several symptoms cause distress to human activities and lifestyle, including fever, breathing difficulty, nausea, chest pain, and cough. In the environment, Zn is generally found in soil and water bodies, where it is introduced through the action of weathering, and release of industrial effluents, respectively. Excessive levels of Zn in these sources can alter soil and aquatic microbial diversity, and can thus affect the bioavailability and absorption of other metals as well. Several Gram-positive and -negative species, such as Bacillus sp., Staphylococcus sp., Streptococcus sp., and Escherichia coli, Pseudomonas sp., Klebsiella sp., and Enterobacter sp., respectively, have been reported to be promising agents of Zn bioremediation. This review intends to present an overview of Zn and its properties, uses, bioavailability, toxicity, as well as the major mechanisms involved in its bioremediation from polluted soil and wastewaters.

Pomegranate (Punica granatum L.) is a non-climacteric and a favorite fruit of tropical, sub-tropical and arid regions of the world. During a survey in autumn 2019, leaf lesions were observed on plants (cv. Kandhari) in different orchards of Muzaffargarh (30°4′27.7572″ N, 71°11′4.7544″ E), a major pomegranate-producing region in Punjab Province. Disease incidence ranged from 17 to 20%. Leaf lesions were initially small (1 to 3 mm in diameter), round, purple or reddish-brown, scattered spots. At later stages, spots increased in size and the centers of mature lesions became dark red or black with fungal sporulation. To isolate the pathogen, samples of leaf (5 × 5 mm) were cut from the junction of diseased and healthy tissue, surface disinfected in 75% alcohol for 30 s, sterilized with 6% sodium hypochlorite for 3 min, washed with sterile distilled water three times, air dried in laminar flow hood, and cultured on potato dextrose agar (PDA). After one week of incubation at 25 ± 2°C with a 12-h photoperiod, fungal colonies developed, which were initially white and became pale yellow with olivaceous green mycelium after 20 days. On PDA, ascomata were olivaceous green, with a papillate ostiole, globose or ovoidal to obovoidal (155 to 220 × 120 to 240 µm, n=50). Terminal and lateral setae were abundant, brown, and tapering toward the tips (4 to 6 µm, n=50). Asci were greenish and lemon-shaped (6 to 8 × 9 to 13.5 µm, n=50). Ascospores were limoniform and olivaceous gray-brown (10 to 11.5 × 7 to 9 µm, n=50). These morphological characteristics were consistent with the morphology of Chaetomium globosum (Lan et al. 2011; Wang et al. 2016). Genomic DNA was extracted from two isolates and identification of the pathogen was confirmed by amplification and sequencing of the internal transcribed spacer region (ITS) and the partial translation elongation factor 1-α (TEF1) gene using ITS1/ITS4 (White et al. 1999) and EF1-983F/EF1-2218R primers (Wang et al. 2016), respectively. The sequences of the PCR products were deposited in GenBank with accession numbers MW522514, MW522352 (ITS), and MW530423, MW530424 (TEF1). BLAST results of the obtained sequences of the ITS and TEF1 genes revealed 100% (513/513 bp) and 99.78% (927/929 bp) similarity with those of C. globosum in GenBank (ITS: KX834823 and KT898637, and TEF1: MG812564 and KC485028). To confirm pathogenicity, inoculum was prepared by harvesting conidia from 10-day-old culture grown in PDA. The surface-disinfected (70% ethyl alcohol, 30 s) leaves of ten 1-year-old seedlings (cv. Kandhari) were sprayed with a spore suspension (1×106 conidia/ml). Leaves of ten seedlings sprayed with sterile distilled water served as controls. All seedlings were covered with plastic bags and placed in a greenhouse at 26°C with 12 h photoperiod. After eight days, symptoms on inoculated leaves were similar to those observed in the orchards; no symptoms were observed on controls. The fungus was reisolated from all symptomatic tissues. C. globosum has been reported on Punica granatum (Guo et al. 2015), Cannabis sativa (Chaffin et al. 2020) and Brassica oleracea (Zhu et al. 2020). This is the first report of C. globosum causing leaf spot on pomegranate in Pakistan. This finding suggests a potential threat to pomegranate production in Pakistan and further studies should focus on effective prevention and control practices of this disease.

In the current study, deterrent assay, contact bioassay, lethal concentration (LC) analysis and gene expression analysis were performed to reveal the repellent or insecticidal potential of M. alternifolia oil against M. persicae. M. alternifolia oil demonstrated an excellent deterrence index (0.8) at 2 g/L after 48 h. The oil demonstrated a pronounced contact mortality rate (72%) at a dose of 4 g/L after 24 h. Probit analysis was performed to estimate LC-values of M. alternifolia oil (40%) against M. persicae (LC30 = 0.115 g/L and LC50 = 0.37 g/L respectively) after 24 h. Furthermore, to probe changes in gene expression due to M. alternifolia oil contact in M. persicae, the expression of HSP 60, FPPS I, OSD, TOL and ANT genes were examined at doses of LC30 and LC50. Four out of the five selected genes—OSD, ANT, HSP 60 and FPPS I—showed upregulation at LC50, whereas, TOL gene showed maximum upregulation expression at LC30. Finally, the major components of M. alternifolia oil (terpinen-4-ol) were docked and MD simulated into the related proteins of the selected genes to explore ligand–protein modes of interactions and changes in gene expression. The results show that M. alternifolia oil has remarkable insecticidal and deterrent effects and also has the ability to affect the reproduction and development in M. persicae by binding to proteins.

This study was aimed to investigate the anticancer potential of Euphorbia milii ( E. milii ) using an exquisite combination of phytopharmacological and advanced computational techniques. The chloroform fraction (Em-C) of E. milii methanol extract showed the highest antioxidant activity (IC 50 : 6.41 ± 0.99 µg/ml) among all studied fractions. Likewise, Em-C also showed significant cytotoxicity (IC 50 : 11.2 ± 0.8 µg/ml) when compared with that of standard compound 5-fluorouracil (5-FU) (IC 50 : 4.22 ± 0.6 µg/ml) against hepatocarcinoma cell line (HepG2). However, in a human cervical cancer cell line (HeLa), Em-C demonstrated a non-significant difference in cytotoxicity (22.1 ± 0.8 µg/ml) when compared with that of 5-FU (IC 50 : 6.87 ± 0.5 µg/ml). Furthermore, Western blot and qRT-PCR analysis revealed that the suppression of HepG2 cells was the consequence of a tremendous decrease in CDK2 and E2F1 protein expression. The GC–MS analysis of Em-C revealed the unique presence of cyclobarbital (CBT) and benzodioxole derivative (BAN) as major constituents. Furthermore, molecular docking of compounds BAN, CBT, and MBT into the binding site of different molecular targets i.e. cyclin dependent kinase 2 (CDK2), thymidylate synthase (TS), caspase 3, BCL2 and topoisomerase II was carried out. Compounds BAN and CBT have demonstrated remarkable binding affinity towards CDK2 and thymidylate synthase, respectively. Molecular dynamic simulation studies have further confirmed the finding of docking analysis, suggesting that CDK2 and TS can act as an attractive molecular target for BAN and CBT, respectively. It can be concluded that these E. milii phytoconstituents (BAN and CBT) may likely be responsible for anti-invasive activity against HepG2 cells.

Sesame (Sesamum indicum L.) commonly known as ‘til’ is the most ancient and widely grown oilseed crop of Pakistan. During 2020, field survey conducted in various research fields of Faisalabad (31°26′00.2″N, 73°04′25.4″E) revealed the occurrence of characteristic leaf blight disease with an incidence of 10 to 13%. The symptoms were characterized by yellow-brown and irregular lesions. At later stages, the lesions expanded and the affected leaves turned grayish to dark-brown and finally became wilted. Symptomatic leaves (both the diseased and healthy tissue) were cut into approximately 2 × 2 mm pieces, surface sterilized with 1% sodium hypochlorite for 30 s, 70% ethanol for 30 s, and finally, three times in sterile distilled water prior to culturing on Potato Dextrose Agar (PDA) and incubated at 25 °C under a 12-h photoperiod for 7 days. To obtain pure cultures, hyphal tips of growing mycelia from leaf tissues were carefully isolated and transferred onto fresh PDA plates. Fungal colonies of 11 isolates were initially white, becoming light to dark-gray. The conidia were black, spherical to subspherical, and single-celled (12 to 14 × 18 to 20 μm) in diameter, which were borne on a hyaline vesicle at the tip of the conidiophore. Further, to identify the pathogen to the species level, genomic DNA was extracted using a modified CTAB protocol described by (Guo et al. 2000). The internal transcribed spacer (ITS) region of the ribosomal DNA and translation elongation factor 1-alpha (TEF1-α) gene were amplified using ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R primer sets (Carbone and Kohn 1999), respectively. The sequences were submitted to GenBank (accession no. MW287214 for ITS and MW325222 for TEF1. The sequences comparison revealed 99% and 100% similarity to multiple sequences of N. sphaerica (GenBank accessions KX834822 and MN995332). On the basis of cultural features, conidial morphology and molecular data, the fungus was identified as Nigrospora sphaerica (Sacc.) Mason (Wang et al. 2017; Chen et al. 2018). To test the pathogenicity, fresh and healthy leaves of ten 6-week-old growth stage sesame plants were spray inoculated with a conidial suspension (105 conidia/ml), collected from a 7-day-old culture on PDA. In addition, 10 plants sprayed with sterile distilled water served as controls. Inoculated plants were covered with polyethylene bags to maintain high humidity and kept at 28°C, and observations were made at regular intervals. After 8-10 days of inoculation, leaves developed blight symptoms similar to those observed on naturally infected leaves, whereas control leaves remained asymptomatic. The pathogen was re-isolated from the inoculated leaves, and its identity was confirmed by morphological and molecular (ITS and TEF1) means, thus fulfilling Koch's postulates. N. sphaerica is distributed on a wide range of hosts and has been reported from different host genera including monocotyledonous and dicotyledonous (Wang et al. 2017). Previously, N. sphaerica has been reported to cause leaf blight of Cunninghamia lanceolata in China (Xu and Liu, 2016). To the best of our knowledge, this is the first report of Nigrospora sphaerica as the causal agent of leaf blight of sesame in Pakistan. Because sesame is an important oilseed crop of Pakistan, appropriate disease management practices should be developed and implemented.