Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants.

Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future.

Exosome/Liposome-like Nanoparticles: New Carriers for CRISPR Genome Editing in Plants.

Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.

Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus.

In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3-5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20-40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60-70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production.

An Outlook on Global Regulatory Landscape for Genome-Edited Crops.

The revolutionary technology of CRISPR/Cas systems and their extraordinary potential to address fundamental questions in every field of biological sciences has led to their developers being awarded the 2020 Nobel Prize for Chemistry. In agriculture, CRISPR/Cas systems have accelerated the development of new crop varieties with improved traits-without the need for transgenes. However, the future of this technology depends on a clear and truly global regulatory framework being developed for these crops. Some CRISPR-edited crops are already on the market, and yet countries and regions are still divided over their legal status. CRISPR editing does not require transgenes, making CRISPR crops more socially acceptable than genetically modified crops, but there is vigorous debate over how to regulate these crops and what precautionary measures are required before they appear on the market. This article reviews intended outcomes and risks arising from the site-directed nuclease CRISPR systems used to improve agricultural crop plant genomes. It examines how various CRISPR system components, and potential concerns associated with CRISPR/Cas, may trigger regulatory oversight of CRISPR-edited crops. The article highlights differences and similarities between GMOs and CRISPR-edited crops, and discusses social and ethical concerns. It outlines the regulatory framework for GMO crops, which many countries also apply to CRISPR-edited crops, and the global regulatory landscape for CRISPR-edited crops. The article concludes with future prospects for CRISPR-edited crops and their products.

Hydrogen peroxide detoxifying enzymes show different activity patterns in host and non-host plant interactions with Magnaporthe oryzae Triticum pathotype.

Wheat blast caused by the hemibiotroph fungal pathogen Magnaporthe oryzae Triticum (MoT) pathotype is a destructive disease of wheat in South America, Bangladesh and Zambia. This study aimed to determine and compare the activities of antioxidant enzymes in susceptible (wheat, maize, barley and swamp rice grass) and resistant (rice) plants when interacting with MoT. The activities of reactive oxygen species-detoxifying enzymes; catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione S-transferase (GST), peroxidase (POX) were increased in all plants in response to MoT inoculation with a few exceptions. Interestingly, an early and very high activity of CAT was observed within 24 h after inoculation in wheat, barley, maize and swamp rice grass with lower H2O2 concentration. In contrast, an early and high accumulation of H2O2 was observed in rice at 48 hai with little CAT activity only at a later stage of MoT inoculation. The activities of APX, GST and POD were also high at an early stage of infection in rice. However, these enzymes activities were very high at a later stage in wheat, barley, maize and swamp rice grass. The activity of GPX gradually decreased with the increase of time in rice. Taken together, our results suggest that late and early inductions of most of the antioxidant enzyme activities occurs in susceptible and resistant plants, respectively. This study demonstrates some insights into physiological responses of host and non-host plants when interacting with the devastating wheat blast fungus MoT, which could be useful for developing blast resistant wheat.

The genomes of the fungal plant pathogens Cladosporium fulvum and Dothistroma septosporum reveal adaptation to different hosts and lifestyles but also signatures of common ancestry.

We sequenced and compared the genomes of the Dothideomycete fungal plant pathogens Cladosporium fulvum (Cfu) (syn. Passalora fulva) and Dothistroma septosporum (Dse) that are closely related phylogenetically, but have different lifestyles and hosts. Although both fungi grow extracellularly in close contact with host mesophyll cells, Cfu is a biotroph infecting tomato, while Dse is a hemibiotroph infecting pine. The genomes of these fungi have a similar set of genes (70% of gene content in both genomes are homologs), but differ significantly in size (Cfu >61.1-Mb; Dse 31.2-Mb), which is mainly due to the difference in repeat content (47.2% in Cfu versus 3.2% in Dse). Recent adaptation to different lifestyles and hosts is suggested by diverged sets of genes. Cfu contains an α-tomatinase gene that we predict might be required for detoxification of tomatine, while this gene is absent in Dse. Many genes encoding secreted proteins are unique to each species and the repeat-rich areas in Cfu are enriched for these species-specific genes. In contrast, conserved genes suggest common host ancestry. Homologs of Cfu effector genes, including Ecp2 and Avr4, are present in Dse and induce a Cf-Ecp2- and Cf-4-mediated hypersensitive response, respectively. Strikingly, genes involved in production of the toxin dothistromin, a likely virulence factor for Dse, are conserved in Cfu, but their expression differs markedly with essentially no expression by Cfu in planta. Likewise, Cfu has a carbohydrate-degrading enzyme catalog that is more similar to that of necrotrophs or hemibiotrophs and a larger pectinolytic gene arsenal than Dse, but many of these genes are not expressed in planta or are pseudogenized. Overall, comparison of their genomes suggests that these closely related plant pathogens had a common ancestral host but since adapted to different hosts and lifestyles by a combination of differentiated gene content, pseudogenization, and gene regulation.

Myconanoparticles: synthesis and their role in phytopathogens management.

Nanotechnology can offer green and eco-friendly alternatives for plant disease management. Apart from being eco-friendly, fungi are used as bio-manufacturing units, which will provide an added benefit in being easy to use, as compared to other microbes. The non-pathogenic nature of some fungal species in combination with the simplicity of production and handling will improve the mass production of silver nanoparticles. Recently, a diverse range of fungi have been screened for their ability to create silver nanoparticles. Mycosynthesis of gold, silver, gold-silver alloy, selenium, tellurium, platinum, palladium, silica, titania, zirconia, quantum dots, usnic acid, magnetite, cadmium telluride and uraninite nanoparticles has also been reported by various researchers. Nanotechnological application in plant pathology is still in the early stages. For example, nanofungicides, nanopesticides and nanoherbicides are being used extensively in agriculture practices. Remote activation and monitoring of intelligent nano-delivery systems can assist agricultural growers of the future to minimize fungicides and pesticides use. Nanoparticle-mediated gene transfer would be useful for improvement of crops resistant to pathogens and pest. This review critically assesses the role of fungi in the synthesis of nanoparticles, the mechanism involved in the synthesis, the effect of different factors on the reduction of metal ions in developing low-cost techniques for the synthesis and recovery of nanoparticles. Moreover, the application of nanoparticles in plant disease control, antimicrobial mechanisms, and nanotoxicity on plant ecosystem and soil microbial communities has also been discussed in detail.

Plant pathogen nanodiagnostic techniques: forthcoming changes?

Plant diseases are among the major factors limiting crop productivity. A first step towards managing a plant disease under greenhouse and field conditions is to correctly identify the pathogen. Current technologies, such as quantitative polymerase chain reaction (Q-PCR), require a relatively large amount of target tissue and rely on multiple assays to accurately identify distinct plant pathogens. The common disadvantage of the traditional diagnostic methods is that they are time consuming and lack high sensitivity. Consequently, developing low-cost methods to improve the accuracy and rapidity of plant pathogens diagnosis is needed. Nanotechnology, nano particles and quantum dots (QDs) have emerged as essential tools for fast detection of a particular biological marker with extreme accuracy. Biosensor, QDs, nanostructured platforms, nanoimaging and nanopore DNA sequencing tools have the potential to raise sensitivity, specificity and speed of the pathogen detection, facilitate high-throughput analysis, and to be used for high-quality monitoring and crop protection. Furthermore, nanodiagnostic kit equipment can easily and quickly detect potential serious plant pathogens, allowing experts to help farmers in the prevention of epidemic diseases. The current review deals with the application of nanotechnology for quicker, more cost-effective and precise diagnostic procedures of plant diseases. Such an accurate technology may help to design a proper integrated disease management system which may modify crop environments to adversely affect crop pathogens.

Special Issue: Microbial Nanotechnology.

Microbial nanotechnology (MN), or microbial nanobiotechnology, is a rapidly expanding research area with the potential to transform various fields, including bioremediation, energy production, medicine, and agriculture [...].

Special Issue: Agricultural Nanotechnology.

Agricultural nanotechnology has considerable promise for addressing global agricultural production/security, biodiversity, and global warming issues. Current trends in publications and patents demonstrate that biotechnology technologies, particularly for crops, are being developed to improve agricultural productivity and disease management. In the current issue, we strongly advocate for the use of biosynthesized nanoparticles from a variety of sources, including plants, agricultural waste, and microbes, as a prerequisite for significant and in-depth study. Nanomaterials offer a wide range of practical uses in agriculture, including nanofertilizers, nanopesticides, nanoherbicides, nanosensors, and smart delivery systems for controlled agrochemical release. Additionally, nano-tools are employed for plant breeding and genetic manipulation. A thorough examination of the physicochemical soil properties of the agricultural fields where nanoparticles will be used will aid in minimizing their impact on plant and soil biota. Finally, and most importantly, we strongly recommend the inclusion of nanotoxicity, legislation, biosafety, and risk assessment as the top priorities when developing regulatory policies to address biosafety concerns. Starting today, thorough efforts must be carried out to advance and develop futuristic work based on recognized knowledge shortages.

Special Issue: Fungal Nanotechnology 2.

Fungal nanotechnology provides techniques useful for molecular and cell biology, medicine, biotechnology, agriculture, veterinary physiology, and reproduction. This technology also has exciting potential applications in pathogen identification and treatment, as well as impressive outcomes in the animal and food systems. Myconanotechnology is a viable option for the synthesis of green nanoparticles because it is simple, affordable, and more environmentally friendly to use fungal resources. Mycosynthesis nanoparticles can be used for various purposes, such as pathogen detection and diagnosis, control, wound healing, drug delivery, cosmetics, food preservation, and textile fabrics, among other applications. They can be applied to a variety of industries, such as agriculture, manufacturing, and medicine. Gaining deeper comprehension of the molecular biology and genetic components underlying the fungal nanobiosynthetic processes is becoming increasingly important. This Special Issue aims to showcase recent advancements in invasive fungal diseases caused by human, animal, plant, and entomopathogenic fungi that are being identified, treated, and treated using antifungal nanotherapy. Utilizing fungus in nanotechnology has several benefits, such as their capacity to create nanoparticles with distinctive characteristics. As an illustration, some fungi can create nanoparticles that are highly stable, biocompatible, and have antibacterial capabilities. Fungal nanoparticles may be used in a variety of industries, including biomedicine, environmental cleanup, and food preservation. Fungal nanotechnology is also a sustainable and environmentally beneficial method. Fungi are an appealing alternative to conventional chemical methods of creating nanoparticles because they are simple to cultivate using affordable substrates and may be cultivated under diverse conditions.

Biosynthesis, Characterization, and Antifungal Activity of Novel Trimetallic Copper Oxide-Selenium-Zinc Oxide Nanoparticles against Some Mucorales Fungi.

Metal nanoparticles are assumed to be a new generation of biologically active materials. The integrations between more than one metal are synergetic multifunctional features. In the current study, trimetallic copper-selenium-zinc oxide nanoparticles (Tri-CSZ NPs) were successfully mycosynthesized using Aspergillus niger through an ecofriendly method for the first time. The biosynthesis of the particles was characterized using physiochemical and topographical analysis. The physiochemical analysis included Fourier transform infrared spectroscopy (FTIR), which affirmed that the biosynthesis of Tri-CSZ NPs relies on the functional groups of fungal filtrates. Additionally, the UV-visible and X-ray diffraction patterns were proposed for the formation of Tri-CSZ NPs; moreover, topography analysis confirmed that the micromorphology of the nanoparticles were similar to a stick, with ends having a tetragonal pyramid shape, and with an average nanosize of about 26.3 ± 5.4 nm. Cytotoxicity results reveled that the Tri-CSZ NPs have no cytotoxicity on the human normal cell line Wi 38 at low concentrations, where the IC50 was 521 µg/mL. Furthermore, the antifungal activity of the Tri-CSZ NPs was evaluated. The antifungal results revealed that the Tri-CSZ NPs have promising antifungal activity against Mucor racemosus, Rhizopus microsporus, Lichtheimia corymbifera, and Syncephalastrum racemosum, where the minimum inhibitory concentrations (MICs) were 1.95, 7.81, 62.5, and 3.9 µg/mL, and the minimum fungicidal concentrations (MFCs) were 250, 62.5, 125, and 1000 µg/mL, respectively. In conclusion, Tri-CSZ NPs were successfully mycosynthesized using A. niger, which have a promising antifungal activity against fungi causing mucormycosis.

Polianthes tuberosa-Mediated Silver Nanoparticles from Flower Extract and Assessment of Their Antibacterial and Anticancer Potential: An In Vitro Approach.

Plant-mediated metallic nanoparticles have beenreported for a diversified range of applications in biological sciences. In the present study, we propose the Polianthes tuberosa flower as a reducing and stabilizing agent for the synthesis of silver nanoparticles (PTAgNPs). The PTAgNPs were exclusively characterized using UV-Visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray diffraction (XRD), atomic force microscopy, zeta potential, and transmission electron microscopy (TEM) studies. In a biological assay, we investigated the antibacterial and anticancer activity of silver nanoparticles in the A431 cell line. The PTAgNPs demonstrated a dose-dependent activity in E. coli and S. aureus, suggesting the bactericidal nature of AgNPs. The PTAgNPs exhibited dose-dependent toxicity in the A431 cell line, with an IC50 of 54.56 µg/mL arresting cell growth at the S phase, as revealed by flow cytometry analysis. The COMET assay revealed 39.9% and 18.15 severities of DNA damage and tail length in the treated cell line, respectively. Fluorescence staining studies indicate that PTAgNPs cause reactive oxygen species (ROS) and trigger apoptosis. This research demonstrates that synthesized silver nanoparticles have a significant effect on inhibiting the growth of melanoma cells and other forms of skin cancer. The results show that these particles can cause apoptosis or cell death in malignant tumor cells. This suggests that they could be used to treat skin cancers without harming normal tissues.

Sustainable biosynthesized bimetallic ZnO@SeO nanoparticles from pomegranate peel extracts: antibacterial, antifungal and anticancer activities.

Sustainable bimetallic nanoparticles (NPs) have attracted particular attention in the past decade. However, the efficiency and environmental concerns are associated with their synthesis and properties optimization. We report herein biosynthesis of bimetallic ZnO@SeO NPs based on green and ecofriendly methods using pomegranate peel extract (PPE). Pyrochemical ultraviolet-visible (UV-vis), Fourier-transform infrared (FTIR) and X-ray diffraction (XRD) spectroscopy as well as TEM and EDX supported successful synthesis. Antibacterial, antifungal, and cytotoxic activities were indicative of biological worth of sustainable bimetallic ZnO@SeO NPs, exhibiting antibacterial activity compared to monometallic ZnO and SeO NPs. The values of Minimum Inhibitory Concentration (MIC) of bimetallic ZnO@SeO NPs toward E. coli, P. aeruginosa, B. subtilis and S. aureus were 3.9, 15.62, 3.9 and 7.81 µg ml-1, respectively. Likewise, a promising antifungal activity against Candida albicans, Aspergillus flavus, A. niger and A. fumigatus was achieved (MICs: 31.25, 1.95, 15.62 and 15.62 µg ml-1, respectively). The cytotoxicity results suggest that bimetallic ZnO@SeO NPs are non-toxic and biomedically safe, evidenced by in vitro anticancer activity against human liver carcinoma (Hep-G2) cell line (with a half-maximal inhibitory concentration (IC50) > 71 µg ml-1). The bimetallic ZnO@SeO NPs successfully biosynthesized using PPE showed a high potential for biomedical engineering.

Zinc Oxide Nanoparticles Biosynthesized by Eriobotrya japonica Leaf Extract: Characterization, Insecticidal and Antibacterial Properties.

Zinc oxide nanoparticles (ZnO-NPs) have gained significant attention in nanotechnology due to their unique properties and potential applications in various fields, including insecticidal and antibacterial activities. The ZnO-NPs were biosynthesized by Eriobotrya japonica leaf extract and characterized by various techniques such as UV-visible (UV-vis) spectrophotometer, X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and zeta potential analysis. The results of SEM revealed that NPs were irregular and spherical-shaped, with a diameter between 5 and 27 nm. Meanwhile, DLS supported that the measured size distributions were 202.8 and 94.7 nm at 11.1° and 90.0°, respectively, which supported the polydisperse nature of NPs, and the corresponding zeta potential was -20.4 mV. The insecticidal activity of the produced ZnO-NPs was determined against the adult stage of coleopteran pests, Sitophilus oryzae (Linnaeus) (Curculionidae) and Tribolium castaneum (Herbst) (Tenebrionidae). The LC50 values of ZnO-NPs against adults of S. oryzae and T. castaneum at 24 h of exposure were 7125.35 and 5642.65 µg/mL, respectively, whereas the LC90 values were 121,824.56 and 66,825.76 µg/mL, respectively. Moreover, the biosynthesized nanoparticles exhibited antibacterial activity against three potato bacterial pathogens, and the size of the inhibition zone was concentration-dependent. The data showed that the inhibition zone size increased with an increase in the concentration of nanoparticles for all bacterial isolates tested. The highest inhibition zone was observed for Ralstonia solanacearum at a concentration of 5 µg/mL, followed by Pectobacterium atrosepticum and P. carotovorum. Eventually, ZnO-NPs could be successfully used as an influential agent in pest management programs against stored-product pests and potato bacterial diseases.

Entomopathogenic Fungi: An Eco-Friendly Synthesis of Sustainable Nanoparticles and Their Nanopesticide Properties.

The agricultural industry could undergo significant changes due to the revolutionary potential of nanotechnology. Nanotechnology has a broad range of possible applications and advantages, including insect pest management using treatments based on nanoparticle insecticides. Conventional techniques, such as integrated pest management, are inadequate, and using chemical pesticides has negative consequences. As a result, nanotechnology would provide ecologically beneficial and effective alternatives for insect pest control. Considering the remarkable traits they exhibit, silver nanoparticles (AgNPs) are recognized as potential prospects in agriculture. Due to their efficiency and great biocompatibility, the utilization of biologically synthesized nanosilver in insect pest control has significantly increased nowadays. Silver nanoparticles have been produced using a wide range of microbes and plants, which is considered an environmentally friendly method. However, among all, entomopathogenic fungi (EPF) have the most potential to be used in the biosynthesis of silver nanoparticles with a variety of properties. Therefore, in this review, different ways to get rid of agricultural pests have been discussed, with a focus on the importance and growing popularity of biosynthesized nanosilver, especially silver nanoparticles made from fungi that kill insects. Finally, the review highlights the need for further studies so that the efficiency of bio-nanosilver could be tested for field application and the exact mode of action of silver nanoparticles against pests can be elucidated, which will eventually be a boon to the agricultural industry for putting a check on pest populations.

Watermelon Rind Mediated Biosynthesis of Bimetallic Selenium-Silver Nanoparticles: Characterization, Antimicrobial and Anticancer Activities.

One of the most hazardous diseases that influences human health globally is microbial infection. Therefore, bimetallic nanoparticles have received much attention for controlling microbial infections in the current decade. In the present study, bimetallic selenium-silver nanoparticles (Se-Ag NPs) were effectively biosynthesized using watermelon rind WR extract through the green technique for the first time. UV-visible spectroscopy, transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy (EDX) methods were used to characterize the produced NPs. The results indicated that the bimetallic Se-Ag NPs had synergistic antimicrobial activity at low concentrations, which helped to reduce the toxicity of Ag NPs after the bimetallic Se-Ag NPs preparation and increase their great potential. Se-Ag NPs with sizes ranging from 18.3 nm to 49.6 nm were detected by TEM. Se-Ag NP surfaces were uniformly visible in the SEM picture. The cytotoxicity of bimetallic Se-Ag NPs was assessed against the Wi38 normal cell line to check their safety, where the IC50 was 168.42 µg/mL. The results showed that bimetallic Se-Ag NPs had antibacterial action against Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Bacillus subtilis, and Staphylococcus aureus with a minimum inhibitory concentration (MIC) of 12.5 to 50 µg/mL. Additionally, bimetallic Se-Ag NPs had promising anticancer activity toward the MCF7 cancerous cell line, where the IC50 was 21.6 µg/mL. In conclusion, bimetallic Se-Ag NPs were biosynthesized for the first time using WR extract, which had strong antibacterial, antifungal and anticancer properties.

Characterization of novel di-, tri-, and tetranucleotide microsatellite primers suitable for genotyping various plant pathogenic fungi with special emphasis on Fusaria and Mycospherella graminicola.

The goals of this investigation were to identify and evaluate the use of polymorphic microsatellite marker (PMM) analysis for molecular typing of seventeen plant pathogenic fungi. Primers for di-, tri-, and tetranucleotide loci were designed directly from the recently published genomic sequence of Mycospherlla graminicola and Fusarium graminearum. A total of 20 new microsatellite primers as easy-to-score markers were developed. Microsatellite primer PCR (MP-PCR) yielded highly reproducible and complex genomic fingerprints, with several bands ranging in size from 200 to 3000 bp. Of the 20 primers tested, only (TAGG)4, (TCC)5 and (CA)7T produced a high number of polymorphic bands from either F. graminearum or F. culmorum. (ATG)5 led to successful amplifications in M. graminicola isolates collected from Germany. Percentage of polymorphic bands among Fusarium species ranged from 9 to 100%. Cluster analysis of banding patterns of the isolates corresponded well to the established species delineations based on morphology and other methods of phylogenetic analysis. The current research demonstrates that the newly designed microsatellite primers are reliable, sensitive and technically simple tools for assaying genetic variability in plant pathogenic fungi.

Pseudomonas indica-Mediated Silver Nanoparticles: Antifungal and Antioxidant Biogenic Tool for Suppressing Mucormycosis Fungi.

Mucormycosis is considered one of the most dangerous invasive fungal diseases. In this study, a facile, green and eco-friendly method was used to biosynthesize silver nanoparticles (AgNPs) using Pseudomonas indica S. Azhar, to combat fungi causing mucormycosis. The biosynthesis of AgNPs was validated by a progressive shift in the color of P. indica filtrate from colorless to brown, as well as the identification of a distinctive absorption peak at 420 nm using UV-vis spectroscopy. Fourier-transform infrared spectroscopy (FTIR) results indicated the existence of bioactive chemicals that are responsible for AgNP production. AgNPs with particle sizes ranging from 2.4 to 53.5 nm were discovered using transmission electron microscopy (TEM). Pattern peaks corresponding to the 111, 200, 220, 311, and 222 planes, which corresponded to face-centered cubic forms of metallic silver, were also discovered using X-ray diffraction (XRD). Moreover, antifungal activity measurements of biosynthesized AgNPs against Rhizopus Microsporus, Mucor racemosus, and Syncephalastrum racemosum were carried out. Results of antifungal activity analysis revealed that the biosynthesized AgNPs exhibited outstanding antifungal activity against all tested fungi at a concentration of 400 µg/mL, where minimum inhibitory concentrations (MIC) were 50, 50, and 100 µg/mL toward R. microsporus, S. racemosum, and M. racemosus respectively. In addition, the biosynthesized AgNPs revealed antioxidant activity, where IC50 was 31 µg/mL when compared to ascorbic acid (0.79 µg/mL). Furthermore, the biosynthesized AgNPs showed no cytotoxicity on the Vero normal cell line. In conclusion, the biosynthesized AgNPs in this study can be used as effective antifungals with safe use, particularly for fungi causing mucormycosis.