The details of all steps involved in the quantification of
biofilm formation in microtiter plates are described. The
presented protocol incorporates information on assessment of
biofilm production by staphylococci, gained both by direct
experience as well as by analysis of methods for assaying
biofilm production. The obtained results should simplify
quantification of biofilm formation in microtiter plates, and
make it more reliable and comparable among different
laboratories.

Quantification of biofilm in microtiter plates: overview oftesting conditions and practical recommendations for assessmentof biofilm production by staphylococci.

Cucumber mosaic cucumovirus (CMV) is a deadly plant virus that results in crop-yield losses with serious economic consequences. In recent years, environmentally friendly components have been developed to manage crop diseases as alternatives to chemical pesticides, including the use of natural compounds such as glycine betaine (GB) and chitosan (CHT), either alone or in combination. In the present study, the leaves of the cucumber plants were foliar-sprayed with GB and CHT—either alone or in combination—to evaluate their ability to induce resistance against CMV. The results showed a significant reduction in disease severity and CMV accumulation in plants treated with GB and CHT, either alone or in combination, compared to untreated plants (challenge control). In every treatment, growth indices, leaf chlorophylls content, phytohormones (i.e., indole acetic acid, gibberellic acid, salicylic acid and jasmonic acid), endogenous osmoprotectants (i.e., proline, soluble sugars and glycine betaine), non-enzymatic antioxidants (i.e., ascorbic acid, glutathione and phenols) and enzymatic antioxidants (i.e., superoxide dismutase, peroxidase, polyphenol oxidase, catalase, lipoxygenase, ascorbate peroxidase, glutathione reductase, chitinase and β-1,3 glucanase) of virus-infected plants were significantly increased. On the other hand, malondialdehyde and abscisic acid contents have been significantly reduced. Based on a gene expression study, all treated plants exhibited increased expression levels of some regulatory defense genes such as PR1 and PAL1. In conclusion, the combination of GB and CHT is the most effective treatment in alleviated virus infection. To our knowledge, this is the first report to demonstrate the induction of systemic resistance against CMV by using GB.

https://www.mdpi.com/1420-3049/25/10/2341/pdf

Improving Regulation of Enzymatic and Non-Enzymatic Antioxidants and Stress-Related Gene Stimulation in Cucumber mosaic cucumovirus-Infected Cucumber Plants Treated with Glycine Betaine, Chitosan and Combination

During the spring of 2019, distinct virus-like symptoms were observed in the Kafr El-Sheikh Governorate in Egypt in naturally infected eggplants. Leaves of affected plants showed interveinal leaf chlorosis, net yellow, chlorotic sectors, mottling, blisters, vein enation, necrotic intervention, and narrowing symptoms. The Alfalfa mosaic virus (AMV) was suspected of to be involved in this disease. Forty plant samples from symptomatic eggplants and 10 leaf samples with no symptoms were collected. The samples were tested by double antibody sandwich ELISA (DAS-ELISA) using AMV-IgG. Six of the 40 symptomatic leaf samples tested positive for AMV, while, DAS-ELISA found no AMV in the 10 leaf samples without symptoms. The AMV Egyptian isolate (AMV-Eggplant-EG) was biologically isolated from the six positive samples tested by DAS-ELISA and from the similar local lesions induced on Chenopodium amaranticolor and then re-inoculated in healthy Solanum melongena as a source of AMV-Eggplant-EG and confirmed by DAS-ELISA. Reverse transcription polymerase chain reaction (RT-PCR) assay with a pair of primers specific for coat protein (CP) encoding RNA 3 of AMV yielded an amplicon of 666 bp from infected plants of Solanum melongena with AMV-Eggplant-EG. The amplified PCR product was cloned and sequenced. Analysis of the AMV-Eggplant-EG sequence revealed 666 nucleotides (nt) of the complete CP gene (translating 221 amino acid (aa) residues). Analysis of phylogeny for nt and deduced aa sequences of the CP gene using the maximum parsimony method clustered AMV-Eggplant-EG in the lineage of Egyptian isolates (shark-EG, mans-EG, CP2-EG, and FRE-EG) with a high bootstrap value of 88% and 92%, respectively. In addition to molecular studies, melatonin (MTL) and salicylic acid (SA) (100 μM) were used to increase the resistance of eggplant to AMV- infection. Foliar spray with MLT and SA caused a significant increase in the morphological criteria (shoot, root length, number of leaves, leaf area, and leaf biomass), chlorophyll and carotenoid content, antioxidant enzymes, and gene expression of some enzymes compared to the infected plants. On the other hand, treatment with MLT and SA reduced the oxidative damage caused by AMV through the reduction of hydrogen peroxide, superoxide anions, hydroxyl radicals, and malondialdehyde. In conclusion, MLT and SA are eco-friendly compounds and can be used as antiviral compounds.

https://www.mdpi.com/2223-7747/10/3/459/pdf

Molecular Characterization of the Alfalfa mosaic virus Infecting Solanum melongena in Egypt and the Control of Its Deleterious Effects with Melatonin and Salicylic Acid.

Approximately 6% of the world's total land area and 20% of the irrigated land are affected by salt stress. Egypt is one such country affected by salt-stress problems. This paper focuses on the role of isolated bacteria, such as Bacillus subtilis and Pseudomonas fluorescens, in alleviating the harmful effects of salt stress. The results show that the irrigation of plants with different concentrations of saline water (0, 75, and 150 mM NaCl) leads to significantly decreased growth criteria, photosynthetic pigments (i.e., chl a, chl b, and carotenoids), and membrane stability index (MSI) values. Moreover, malondialdehyde (MDA), glutathione content, endogenous proline, the antioxidant defense system, 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase (ACS), ACC oxidase (ACO), and Na+ content were significantly increased under NaCl-stress exposure. On the contrary, treatment with endophytic bacteria significantly increased the resistance of pea plants to salt stress by increasing the enzymatic antioxidant defenses (i.e., superoxide dismutase, catalase, peroxidase, and glutathione reductase), non-enzymatic antioxidant defenses (i.e., glutathione), osmolyte substances such as proline, and antioxidant enzyme gene expression. As a result, endophytic bacteria's use was significantly higher compared to control values for indole-3-acetic acid (IAA), gibberellic acid GA3, MSI, and photosynthetic pigments. The use of endophytic bacteria significantly decreased Na+ accumulation while, at the same time, promoting K+ uptake. In conclusion, the induction of endophytic bacterium-induced salt tolerance in pea plants depends primarily on the effect of endophytic bacteria on osmoregulation, the antioxidant capacity, and ion uptake adjustment by limiting the uptake of Na+ and, alternatively, increasing the accumulation of K+ in plant tissue.

ACC deaminase containing endophytic bacteria ameliorate salt stress in Pisum sativum through reduced oxidative damage and induction of antioxidative defense systems.

Tomato mosaic virus (ToMV) is one of the economically damageable Tobamovirus infecting the tomato in Egypt that has caused significant losses. It is therefore of great interest to trigger systemic resistance to ToMV. In this endeavor, we aimed to explore the capacity of ZnO-NPs (zinc oxide nanoparticles) to trigger tomato plant resistance against ToMV. Effects of ZnO-NPs on tomato (Solanum lycopersicum L.) growth indices and antioxidant defense system activity under ToMV stress were investigated. Noticeably that treatment with ZnO-NPs showed remarkably increased growth indices, photosynthetic attributes, and enzymatic and non-enzymatic antioxidants compared to the challenge control. Interestingly, oxidative damage caused by ToMV was reduced by reducing malondialdehyde, H2O2, and O2 levels. Overall, ZnO-NPs offer a safe and economic antiviral agent against ToMV.

https://www.mdpi.com/1420-3049/26/5/1337/pdf

Ameliorating the Adverse Effects of Tomato mosaic tobamovirus Infecting Tomato Plants in Egypt by Boosting Immunity in Tomato Plants Using Zinc Oxide Nanoparticles.

Synthetic antimicrobials have a negative impact on food quality and consumer health, which is why natural antimicrobials are urgently needed. Coagulase-negative staphylococci (CoNS) has gained considerable importance for food poisoning and infection in humans and animals, particularly in biofilms. As a result, this study was conducted to control the CoNS isolated from food samples in Egypt. CoNS isolates were selected on the basis of their antibiotic susceptibility profiles and their biofilm-associated behavior. In this context, a total of 29 different bacteriophages were isolated and, in particular, lytic phages (6 isolates) were selected. The host range and physiological parameters of the lytic phages have been studied. Electron microscopy images showed that lytic phages were members of the families Myoviridae (CoNShP-1, CoNShP-3, and CoNSeP-2 isolates) and Siphoviridae (CoNShP-2, CoNSsP-1, and CoNSeP-1 isolates). CoNShP-1, CoNShP-2, and CoNShP-3 were found to be virulent to Staphylococcus haemolyticus, CoNSsP-1 to Staphylococcus saprophyticus and CoNSeP-1 and CoNSeP-2 to Staphylococcus epidermidis. Interestingly, the CoNShP-exhibited a typical polyvalent behavior, where not only lysis CoNS, but also other genera include Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), Bacillus cereus and Bacillus subtilis. In addition, CoNShP-3 phage showed high stability at different temperatures and pH levels. Indeed, CoNShP-3 phage showed an antibiofilm effect against Staphylococcus epidermidis CFS79 and Staphylococcus haemolyticus CFS43, respectively, while Staphylococcus saprophyticus CFS28 biofilm was completely removed. Finally, CoNShP-3 phage demonstrated a high preservative efficacy over short and long periods of storage against inoculated CoNS in chicken breast sections. In conclusion, this study highlights the control of CoNS pathogens using a polyvalent lytic phage as a natural antibacterial and antibiofilm agent from a food safety perspective.

/pdf/polyvalent-phage-conshp-3-as-a-natural-antimicrobial-agent-2oxsxmgptr.pdf

Polyvalent Phage CoNShP-3 as a Natural Antimicrobial Agent Showing Lytic and Antibiofilm Activities against Antibiotic-Resistant Coagulase-Negative Staphylococci Strains

Broad bean mottle virus (BBMV) infects a wide range of hosts, resulting in significant production reductions. The lack of adequate and effective control methods involves implementing novel BBMV control strategies. Herein, we demonstrate the effect of different potassium concentrations (20, 40, and 60 mM) against BBMV in broad bean plants. Potassium could control BBMV infection in broad bean by inhibiting the virus. In addition, infection with BBMV caused a significant decrease in morphological criteria, SPDA, photosynthetic characteristics, phytohormones, and mineral content in broad bean leaves compared to control plants. The levels of reactive oxygen species (ROS) (hydrogen peroxide, hydroxyl radical, and oxygen anion) and ROS scavenging enzymes (catalase, superoxide dismutase, peroxidase, phenylaniline ammonia-lyase, chitinase, and 1,3 - glucanase) increased significantly in plants inoculated with BBMV alone or in the presence of K+. In addition, proline and phenolic compounds increased significantly after being infected with BBMV. In conclusion, treatment with a high potassium concentration (60 mM) alleviates the adverse effect of BBMV on broad bean plants by boosting secondary metabolites, phytohormones, and enzymatic antioxidants.

https://www.notulaebotanicae.ro/index.php/nbha/article/download/12654/9345

A polishing the harmful effects of Broad Bean Mottle Virus infecting broad bean plants by enhancing the immunity using different potassium concentrations

Cucumber production exposed to huge losses in Egypt and worldwide due to infection with Cucumber mosaic Cucumovirus (CMV). The current study focus on the impact of two Egyptian CMV isolates on metabolic and oxidative activities in cucumber plants. Two CMV isolates was obtained from cucurbitaceous crops and confirmed by DASELISA. Inoculated cucumber plants by infectious crude sap with two CMV isolates showed difference in external symptoms. The metabolic and biochemical components of cucumber plants were affected with two CMV isolates. The present study provided that, CMV infection caused a significant reduction in both photopigments and biochemical components while bioactive components (proline and phenol) and oxidative enzymes (POX, SOD, PPO and CAT) were increased significantly compared healthy ones. It was found that cucumberCMV isolate was more effective in metabolic and biochemical than Squash-CMV isolate.

https://ajs.journals.ekb.eg/article_31670_e2350cd95b90e20565472490d52b1817.pdf

Metabolic changes of cucumber plants due to two cmv egyptian isolates

New and natural antimicrobials as an alternative control system are now an urgent need to overcome stubborn bacterial infections. Salmonella Typhimurium has become the most frequent serovar responsible for salmonellosis in humans around the world. The high antimicrobial resistance and biofilm production make this pathogen more dangerous. We aimed to isolate a broad lytic phage to prevent Salmonella infection and reduce its biofilms. Using Salmonella Typhimurium (ST-4) as a host, seven phages were isolated, and only three phages showed clear lytic plaques, two members of the Siphoviridae family (vB_STS-1 and vB_STS-3) and one of the Myoviridae family (vB_STM-2). The vB_STM-2 phage was the most potent broad lytic phage, infecting 100% of tested Salmonella Typhimurium serovars and non-Salmonella strains. Additionally, the vB_STM-2 phage was thermostable at −20 to 55 °C up to 24 h, while at 65 and 75 °C, a significant (p < 0.05) titer reduction was observed after 7 days. Moreover, the phage seemed to be stable at different pHs (4–11) after one to twelve hours (hrs), while increasing the time made the phage more sensitive to the alkaline medium rather than the acidic medium. Interestingly, the vB_STM-2 phage had the capacity to diminish or eradicate the biofilms of tested Salmonella Typhimurium, e.g., ST-4, ST-19, ST-30, ST-37, ST-45 and ST-49 by 81.2%, 76.4%, 43.6%, 41%, 39.8% and 93.4%, respectively, at a titer concentration of 106 PFU/mL. Eventually, the vB_STM-2 phage showed significant (p < 0.05) efficacy in the elimination of Salmonella Typhimurium (ST-4) from contaminated chicken breasts at both storage periods with high titer stability. The Salmonella count showed a severe decline from 7.00 ± 0.63 log10 CFU/cm2 to 0.88 ± 0.17 log10 CFU/cm2 on the seventh day of the short-term storage, and from 5.13 ± 0.44 log10 CFU/cm2 to 1.10 ± 0.12 log10 CFU/cm2 on day 27 of the long-term assay. In both periods, the phage titers remained stable, with insignificant (p < 0.05) loss. Therefore, this phage is considered a prime candidate to combat multi-drug-resistant Salmonella Typhimurium and its biofilms.

Discovery of Polyvalent Myovirus (vB_STM-2) Phage as a Natural Antimicrobial System to Lysis and Biofilm Removal of Salmonella Typhimurium Isolates from Various Food Sources

Ehab E. Refaey

Papers

Molecular Characterization of the Alfalfa mosaic virus Infecting Solanum melongena in Egypt and the Control of Its Deleterious Effects with Melatonin and Salicylic Acid.

Polyvalent Phage CoNShP-3 as a Natural Antimicrobial Agent Showing Lytic and Antibiofilm Activities against Antibiotic-Resistant Coagulase-Negative Staphylococci Strains

A polishing the harmful effects of Broad Bean Mottle Virus infecting broad bean plants by enhancing the immunity using different potassium concentrations

Metabolic changes of cucumber plants due to two cmv egyptian isolates

Discovery of Polyvalent Myovirus (vB_STM-2) Phage as a Natural Antimicrobial System to Lysis and Biofilm Removal of Salmonella Typhimurium Isolates from Various Food Sources