Bacterial Cell Suspensions Research Articles

Exploring the effects of entomopathogenic nematode symbiotic bacteria and their cell free filtrates on the tomato leafminer Tuta absoluta and its predator Nesidiocoris tenuis

The use of biocontrol agents, such as predators and entomopathogenic nematodes, is a promising approach for the effective control of the tomato leafminer Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidaean), an oligophagous insect feeding mainly on Solanaceae species and a major pest of field- and greenhouse-grown tomatoes globally. In this context, the effects of two entomopathogenic nematode species Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora (Poinar) (Rhabditida: Heterorhabditidae), as well as their respective bacterial symbionts, Xenorhabdus nematophila and Photorhabdus luminescens (Enterobacterales: Morganelaceae), which were applied as bacterial cell suspensions and as crude cell-free liquid filtrates on T. absoluta larvae, were investigated. The results showed that of all treatments, the nematodes S. carpocapsae and H. bacteriophora were the most effective, causing up to 98 % mortality of T. absoluta larvae. Regarding bacteria and their filtrates, the bacterium X. nematophila was the most effective (69 % mortality in young larvae), while P. luminescens and both bacterial filtrates showed similar potency (ca. 48–55 % mortality in young larvae). To achieve a holistic approach of controlling this important pest, the impact of these factors on the beneficial predator Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) was also studied. The results demonstrated that although nematodes and especially S. carpocapsae, caused significant mortality on N. tenuis (87 %), the bacterial cell suspensions of X. nematophila and P. luminescens and crude cell-free liquid filtrates had minimum impact on this beneficial predator (∼11–30 % mortality).

SecMet-FISH: labeling, visualization, and enumeration of secondary metabolite producing microorganisms.

Our understanding of the role of secondary metabolites in microbial communities is challenged by intrinsic limitations of culturing bacteria under laboratory conditions and hence cultivation independent approaches are needed. Here, we present a protocol termed Secondary Metabolite FISH (SecMet-FISH), combining advantages of gene-targeted fluorescence in situ hybridization (geneFISH) with in-solution methods (in-solution FISH) to detect and quantify cells based on their genetic capacity to produce secondary metabolites. The approach capitalizes on the conserved nature of biosynthetic gene clusters (BGCs) encoding adenylation (AD) and ketosynthase (KS) domains, and thus selectively targets the genetic basis of non-ribosomal peptide and polyketide biosynthesis. The concept relies on the generation of amplicon pools using degenerate primers broadly targeting AD and KS domains followed by fluorescent labeling, detection, and quantification. Initially, we obtained AD and KS amplicons from Pseuodoalteromonas rubra, which allowed us to successfully label and visualize BGCs within P. rubra cells, demonstrating the feasibility of SecMet-FISH. Next, we adapted the protocol and optimized it for hybridization in both Gram-negative and Gram-positive bacterial cell suspensions, enabling high-throughput single cell analysis by flow cytometry. Ultimately, we used SecMet-FISH to successfully distinguish secondary metabolite producers from non-producers in a five-member synthetic community.

Efficiency of bacterial isolates on suppressing brown spot in rice plants

AbstractMany fungicides can control brown spot, caused by Bipolaris oryzae in rice (Oryza sativa L.), although searching for beneficial microorganisms for biocontrol will improve sustainable integrated pest management. The aim of this investigation was the selection of natural suppressors of the brown spot disease in rice. We performed biochemical (E1), molecular antagonism (E2) and severity suppression (E3) studies under laboratory and greenhouse conditions. The E1, E2 and E3 assays were performed with 21 isolates. The experimental design was completely randomized with three replicates. E1 characterized all 21 isolates for the production of extracellular enzymes, siderophores, biofilm, indole‐3‐acetic acid (IAA) and phosphate solubilization. E2 was conducted to test the in vitro antagonism between the 21 isolates and B. oryzae, and E3 was conducted under greenhouse conditions, where seeds of the rice cultivars BRS‐Primavera and Esmeralda were sown in plastic trays containing 3 kg of fertilized soil. After 45 days, rice leaves were sprayed and inoculated with a mixed suspension of bacterial cells (1 × 108 CFU) and B. oryzae. All of the 21 bacterial isolates produced lipase, N‐fixation, siderophore, IAA, biofilm and solubilized phosphorus. Bacillus cereus, Priestia megaterium, Stenotrophomonas nitritireducens, Serratia marcescens and Serratia nematodiphila reduced radial colony growth of B. oryzae and suppressed brown spot severity by up to 90%. This indicates the potential of these isolates for biocontrol of brown spot in rice as a component of rice disease management.

The MATH test. A three-phase assay?

This study aimed to investigating the possible interference caused by glass test tubes on the quantification of bacterial adhesion to hydrocarbons by the MATH test. The adhesion of four bacteria to hexadecane and to glass test tubes was evaluated employing different suspending polar phases. The role of the ionic strength of the polar phase regarding adhesion to glassware was investigated. Within the conditions studied, Gram-positive bacteria adhered to both the test tube and the hydrocarbon regardless of the polar phase employed; meanwhile, Escherichia coli ATCC 25922 did not attach to either one. The capacity of the studied microorganisms to adhere to glassware was associated with their electron-donor properties. The ionic strength of the suspending media altered the patterns of adhesion to glass in a strain-specific manner by defining the magnitude of electrostatic repulsion observed between bacteria and the glass surface. This research demonstrated that glass test tubes may interact with suspended bacterial cells during the MATH test under specific conditions, which may lead to overestimating the percentage of adhesion to hydrocarbons and, thus, to erroneous values of cell surface hydrophobicity.

Seleção de isolados bacterianos antagonistas e supressores de brusone em plantas de arroz

ABSTRACT Grain yields of rice (Oryza sativa) are affected globally by rice blast (Magnaporthe oryzae). The main objective of this study was to identify isolates of rhizobacterial antagonists of M. oryzae (BRM10781) and screen the most effective isolates for suppressing rice blast under greenhouse conditions. Two assays (E1 and E2) were performed with 22 treatments in a completely randomized design with three replicates. E1 investigated in vitro antagonism between 21 isolates and M. oryzae under laboratory conditions. The E2 experiments were conducted under greenhouse conditions, with rice cultivar BRS Primavera seeds in plastic trays containing 3 kg of fertilized soil. After 21 days, the rice leaves were spray-inoculated with a bacterial cell suspension (1 × 108 CFU) and M. oryzae (3 × 105 conidia.mL-1) or with water (absolute control). Seven isolates, Serratia marcescens (BRM65918, BRM65923, BRM65926, and BRM63532), Bacillus cereus (BRM65919), Stenotrophomonas nitritireducens (BRM65917), and Priestia megaterium (BRM65929), reduced radial growth of M. oryzae colonies from 80.26 to 77.33%. The best leaf blast severity reducers were Pseudomonas nitroreducens (BRM32112), B. thuringiensis (BRM65928), P. megaterium (BRM65916), S. marcescens (BRM65918), S. nematodiphila (BRM63522), and Enterobacter hormaechei (BRM65925), varying from 97 to 95% respectively. The isolate BRM65918 (S. marcescens) showed the best efficiency for both antagonism and disease suppression, indicating its potential as a bioproduct for the biocontrol of rice blast in rice plants.

Enzyme characterization of lactic acid bacteria isolated from duck excreta.

The production of lignocellulosic biomass waste in the agricultural sector of Indonesia is quite high annually. Utilization of lignocellulosic biomass waste through fermentation technology can be used as feed and biofuel. Fermentation technology requires the involvement of micro-organisms such as bacteria (lactic acid bacteria or LAB). LABs can be isolated from various sources, such as duck excreta. However, there have not been many reports of LAB from duck excreta. The present study aimed to characterize LAB enzymes isolated from duck excreta and obtain LAB enzymes with superior fermentation properties. A total of 11 LAB cultures obtained from duck excreta in Yogyakarta, Indonesia, were tested. Enzyme characterization of each LAB was performed using the API ZYM kit (BioMérieux, Marcy-I'Etoile, France). The bacterial cell suspension was dropped onto the API ZYM™ cupule using a pipette and incubated for 4 h at 37°C. After incubation, ZYM A and ZYM B were dripped onto the API ZYM cupule, and color changes were observed for approximately 10 s under a strong light source. Esterase activity was moderate for all LABs. The activity of α-chymotrypsin, β-glucuronidase, α-fucosidase, and α-mannosidase was not observed in a total of 10 LAB. The phosphohydrolase and amino peptidase enzyme activity of seven LABs was strong. Only six LAB samples showed protease activity. The glycosyl hydrolase (GH) activity was observed in a total of 8 LAB, while the activity of 2 LAB was strong (Lactococcus lactis subsp. lactis K5 and Lactobacillus brevis M4A). A total of 2 LABs have superior properties. L. lactis subsp. lactis K5 and L. brevis M4A have a high potential to be used in fermentation. They have the potential for further research, such as their effectiveness in fermentation, lignocellulose hydrolysis, feed additives, molecular characterization to detect specific enzymes, and their specific activities.

Влияние ионизирующего излучения на активность антиоксидантных ферментов штамма Escherichia coli «ПЛ-6» при многократном облучении

The article presents the analysis of enzymatic activity of Escherichia coli “PL-6” cells following multiple gamma irradiation with increasing doses. For irradiation the stationary gamma ray unit “Researcher” (Co-60) with the dose rate of 36.7 Gy/min was used. Radiation doses to the bacterial cells increased from 3.5 to 10 kGy in 0.5 kGy increment, and from 10 to 30 kGy in 5 kGy increment. Unirradiated suspended bacterial cells were used as control. The irradiated cells enzymatic activity was estimated with the spectrophotometric method on the "MultiSkan Go" device (Thermo Fisher Scientific, Finland). The results of the study demonstrate the increase in the E. coli ”PL-6” cells radioresistance caused by gamma irradiation of the cells with gradually increased doses. Exposure of the bacteria to a higher gamma radiation dose caused decrease in the number of viable cells from 10Е+9 CFU/ml in the control culture sample to 10Е+2 CFU/ml after irradiation at a dose of 30 kGy. The most radiation-resistant cells were selected from cells undergone successive multiple irradiation with constantly increasing dose of ionizing radiation. The enzymes of the antioxidant system in irradiated cells exhibit increased specific activity as compared to the enzymes in control, unirradiated, cells. The activity of enzymes increases with an increase in the radiation dose. Catalase activity increased after multiple irradiation by 5 times and peroxidase activity increased by 4 times. To compare the results in the groups, the Student's t-test was applied at p<0.05.

Ex Vivo Fermentation of Hay and Corn by Rumen Bacteria from Cattle and Sheep

Sheep are often utilized as a model ruminant, despite a lack of functional comparisons of rumen bacterial communities and responses during dietary transitions between sheep and cattle. Therefore, an ex vivo study was conducted to evaluate species differences. Rumen fluid was obtained from hay-fed sheep and cattle (n = 3 species−1). Mixed bacterial cell suspensions in buffered media containing 3% w/v ground hay, corn, or combinations (2:1, 1:2) of substrates were incubated (24 h; 39 °C). Suspension pH, lactate, volatile fatty acids (VFA), and digestibility were assessed, functional guilds enumerated, and amylolytic bacteria isolated. Lactate was fully utilized in all hay incubations, and pH did not differ between species (p > 0.75). In contrast, digestibility, lactate accumulation, and pH decline were greater in bovine suspensions fermenting corn (p < 0.01). Streptococcus bovis was the predominant bacteria regardless of species, but total amylolytic bacteria were 10-fold greater in bovine suspensions (p < 0.01). Lactate-utilizing bacteria were 1000-fold greater in bovine than ovine suspensions (p < 0.01). However, total VFA did not differ between species (p > 0.28). Overall, these results demonstrate differential feed utilization capacities in rumen microbial communities of sheep and cattle as well as potential differences in rumen acidosis susceptibility.

Improved PCR for Detection of Xanthomonas euvesicatoria pv. perforans in tomato seeds

Abstract The detection efficiency of the PCR methods for Xanthomonas euvesicatoria pv. perforans (Xep) was determined. Xep specific primer set; HpaF-f/HpaF-r was compared with the previously reported Bs-XpF/Bs-XpR primers in multiplex PCR reaction and singleplex PCR for detection of Xep were tested. The improvement of PCR amplification efficacy was investigated by adding PCR additives or either 5% DMSO or 1 M betaine. Adding both PCR additives gave the same lowest detection limit of Xep detection in both multiplex and singleplex PCR at 100 fg. However, multiplex PCR gave no equal amplification rate for both sets of primers and also showed cross-amplification to X. vesicatoria. Then singleplex PCR with both PCR additives was further tested in a tomato seed lot. The artificially inoculated tomato seeds with various concentrations of Xep from 1.05 x 105 to 101 CFU/ml were tested with singleplex PCR. The lowest detection limit was 101 CFU/ml of the 5 replications of each inoculated seed. One artificially inoculated tomato seed from each bacterial cell suspension was mixed with 2,000 healthy seeds making 0.05% (1/2,000) contaminated tomato seeds sample and further tested with singleplex PCR, results showed the detection limit of 101 CFU/ml in 0.05% Xep contaminated seed lot. Keywords: PCR additives, Seed, Bacterial leaf spot, Tomato, Conventional PCR

Bacillus velezensis Strain HN-Q-8 Induced Resistance to Alternaria solani and Stimulated Growth of Potato Plant.

Bacillus velezensis HN-Q-8, isolated in our previous study, has an antagonistic effect on Alternaria solani. After being pretreated with a fermentation liquid with HN-Q-8 bacterial cell suspensions, the potato leaves inoculated with A. solani displayed smaller lesion areas and less yellowing than the controls. Interestingly, the activity levels of superoxide dismutase, peroxidase, and catalase in potato seedlings were enhanced by the addition of the fermentation liquid with bacterial cells. Additionally, the overexpression of key genes related to induced resistance in the Jasmonate/Ethylene pathway was activated by the addition of the fermentation liquid, suggesting that the HN-Q-8 strain induced resistance to potato early blight. In addition, our laboratory and field experiments showed that the HN-Q-8 strain can promote potato seedling growth and significantly increase tuber yield. The root activity and chlorophyll content of potato seedlings were significantly increased along with the levels of indole acetic acid, gibberellic acid 3, and abscisic acid upon addition of the HN-Q-8 strain. The fermentation liquid with bacterial cells was more efficient in inducing disease resistance and promoting growth than bacterial cell suspensions alone or the fermentation liquid without bacterial cells. Thus, the B. velezensis HN-Q-8 strain is an effective bacterial biocontrol agent, augmenting the options available for potato cultivation.

Bactericidal Activity of Graphene Oxide Tests for Selected Microorganisms.

The aim of this study was to determine the bactericidal potential of graphene oxide (GO) in contact with four species of bacteria: E. coli, S. mutans, S. aureus and E. faecalis. Bacterial cell suspensions of each species were incubated in a medium containing GO, with incubation times of 5, 10, 30 and 60 min, at final concentrations of 50, 100, 200, 300 and 500 μg/mL. The cytotoxicity of GO was evaluated using live/dead staining. The results were recorded using a BD Accuri C6 flow cytofluorimeter. Obtained data were analyzed using BD CSampler software. A significant bacteria viability reduction was noted in all GO-containing samples. The antibacterial properties of GO were strongly influenced by GO concentration and incubation time. The highest bactericidal activity was observed at concentrations of 300 and 500 μg/mL for all incubation times (5, 10, 30 and 60 min). The highest antimicrobial potential was observed for E. coli: after 60 min, the mortality rate was 94% at 300 µg/mL GO and 96% at 500 µg/mL GO; the lowest was found for S. aureus-49% (300 µg/mL) and 55% (500 µg/mL).

91Ex Vivo Fermentation of Corn and hay by Rumen Bacteria from Sheep and Cattle

Abstract Data from cattle and sheep studies are often utilized interchangeably to make inferences regarding ruminant nutrition and physiology, but few studies have directly compared rumen microbiological responses. The objective of this study was to evaluate species differences in ex vivo fermentation of concentrates and forages. Rumen fluid was obtained via oral intubation from Holstein steers (n = 3; age: 212 ± 8 d) and Polypay ram lambs (n = 3; age; 34 ± d) fed a 100% tall fescue (TF) hay diet ad libitum. Mixed bacterial cell suspensions in buffered media contained the following feed substrate treatments (3% wt/vol): TF hay (HY), 2:1 TF hay-to-corn (HC), 2:1 corn-to-TF hay (CH), and corn (CN). Samples were collected after 2, 4, 8, and 24 h of incubation (39°C) for analysis of pH and lactate. Data were analyzed by PROC MIXED in SAS (v. 9.4, SAS Inst. Inc., Cary, NC). The pH differed by species (P = 0.004), treatment (P = 0.005), and time (P < 0.001), with a species by treatment by time interaction (P = 0.001). The pH at 2 h was 6.61 ± 0.02 regardless of species or treatment, and pH declined by 24 h for both steer and ram rumen cell suspensions in all treatments (P < 0.001). Final pH at 24 h did not differ between species for HY (6.15 ± 0.03), but for all treatments including corn, pH was lower in steer rumen cell suspensions (HC: steer – 5.86, ram – 6.06; CH: steer – 5.59, ram – 5.98; CN: steer – 5.26, ram – 5.91 ± 0.05; P ≤ 0.004). Lactate accumulation differed by species (P = 0.038), treatment (P = 0.048), and time (P < 0.0001), with a species by treatment by time interaction (P < 0.001). Lactate at 8 h did not differ between species for HY (steer:3.06, ram: 9.22 ±.84 mmolּ L-1), but was greater in steer than in ram cell suspensions fermenting HC (steer: 12.64; ram: 6.39 ±.84 mmolּ L-1), CH (steer: 13.44; ram: 3.21 ±.84 mmolּ L-1), and CN (steer:3.63; ram:.10 ±.84 mmolּ L-1; P ≤ 0.023). By 24 h, lactate accumulation was minimal for HY, HC, and CH (≤ 1.50 mmolּ L-1) and did not differ between species (P ≥ 0.657). However, lactate at 24 h was greater in steer rumen cell suspensions fermenting CN (steer: 25.78; ram: 182 ± 1.84 mmolּ L-1; P < 0.001). These results indicate that the rumen microbial community of sheep and cattle may differ both in composition and function resulting in different capacities to utilize common dietary feed ingredients.

Influence of Nutrient Medium Composition for Culturing Bacteria and the Dose of <i>B. subtilis</i> 10-4 Biologics on Growth Indicators and Productivity of Wheat Plants

Inoculation of crop seeds with selective strains of growth-stimulating bacteria is an environmentally friendly, low-cost way to increase their yield, but may depend on some biotechnological factors affecting their physiological activity. These include the conditions for the cultivation of bacteria and the dose of the drug. The work analyzed the effectiveness of inoculation of Bashkir spring wheat with 28 different preparations of the Bacillus subtilis strain 10-4: 1 - an aqueous suspension of bacterial cells washed off potato-glucose agar (PGA), 2 - a liquid culture containing cells with exometabolites obtained by cultivating bacteria in potato- glucose broth (PGB), 3 - liquid culture of bacterial cells grown in legume-glucose broth (LGB). At the same time, the growth-stimulating effect of applying a high dose (108 cells/ml) and a low dose (104-105 cells/ml) of bacteria in the inoculum was evaluated. When comparing the preparations obtained from potato-glucose medium, it was revealed that the positive growth effect (an increase in the number of wheat plant roots) was caused only by a high dose of the drug cells in the PGA and both doses of the drug in the PGB, but a high dose of the drug in the PGB inhibited seed germination up to 74% (in the control 100%). When comparing the preparations obtained during the cultivation of bacteria in PGB and LGB, it was revealed that the best growth effect (total root length and shoot height) was greater than control in plants inoculated with a small dose of the drug in PGB and a large dose of the drug in LGB. The stimulating effect of a small dose of the drug in LGB was less and extended only to the root system. The results of laboratory experiments coincided with the effectiveness of the studied drugs in the field. The use of a drug obtained in BGO and administered at a dose of 108 cells / ml, and a drug obtained in PGB and administered at a dose of 105 cells/ml, increased grain yield by 1.6-1.7 times with a decrease in the intensity of leaf-stem diseases by 32 and 11% compared with the untreated control. The drug obtained in LGB and introduced in a small dose did not provide either an increase in yield or a protective effect compared to the control. Possible causes of the action of B. subtilis 10-4 strain preparations related to the production of phytohormones by it, depending on the composition of the medium, and with different adaptive capacity of bacteria when cultured on nitrogen-rich and less nitrogen-rich media are discussed.

The anti-adherence activity and bactericidal effect of GO against Streptococcus mutans from Iraqi dental patients.

The high rate of microbes and their biological activity in the patient's mouth is a concern in the domains of dental caries and periodontal disease. The study aimed to shed light on the relationship between graphene oxide's nanoparticles (nGOs) antimicrobial properties and the growth of dental pathogenic bacteria. The forty swab samples were frequently collected from the patient's cavity mouth between November 2019 and January 2020, from patients who visited dentist clinics in Baghdad by taking swabs from mouth cavities with various dental caries with two age groups (5-17) and (18-60) from male and female to streaking them on Brain-Heart Infusion (BHI) agar, then identified by re-streaking on Mitis Salivarius Bacitracin (MSB) agar. All isolates were confirmed as Streptococcus mutans after API 20 Strep method. As well as the Colony Forming Units (CFU) were then determined after diluting the bacterial cell suspensions to obtain cell samples containing 1.5 × 108CFU/ ml. The collagen-binding adhesin (cnm) and glucosyltransferases (gtf) of S. mutans genes were identified using polymerase chain reaction (PCR) method before and after exposure to the nGOs, which were prepared in different pulse laser energy (500, 600, and 700mJ) with presence and absence of the magnetic field, and the data have been analyzing. After counting the CFU, the nGOs shows high effectiveness inhibiting the growth of S. mutans. This research provides definitive answers about the relationship between nGOs, antibacterial caries, and periodontal disease.

P917 Intestinal complement interacts with commensal microbiota and provides pattern recognition in Inflammatory Bowel Disease

Abstract Background The complement system is a central humoral part of innate immunity and provides essential pattern recognition capabilities as well as effector functions such as opsonisation and target cell lysis. Its prominent role in infection and immunity is undisputed but its involvement in the pathogenesis of inflammatory bowel diseases remains less clear. Published data point to a role of complement within the intestinal lumen supporting clearance of pathogens1. Methods Expression of complement proteins within the intestinal epithelium was quantified by real-time qPCR on lesional and non-lesional biopsy samples from patients with IBD and healthy controls. The concentration of complement proteins was measured in fecal supernatants using ELISA. Flow cytometry of fecal bacterial single cell suspensions was used to assess complement (C3) and antibody (IgA) opsonisation status. Finally, cytometrically distinct populations were isolated by fluorescence-assisted cell sorting (FACS) and microbial community composition was investigated using 16S rDNA sequencing. Prediction of functional profiles from amplicon data was performed using Tax4Fun2. Results Complement expression in intestinal tissue was increased during active inflammation but certain targets were significantly less expressed in lesional mucosal sites (e.g. C6, C7). Free complement proteins in fecal supernatants were mostly undetectable by ELISA. In contrast, complement opsonisation was verified on fecal bacteria using flow cytometry. The degree of complement deposited on bacterial surfaces was significantly correlated with fecal calprotectin (p < 0.01). The percentage of complement-opsonized bacteria ranged from 0 to 5.3 % and did not correlate with intestinal inflammation while percentages of IgA opsonisation were generally higher (0.4 - 90.1%) and increased with fecal calprotectin (p < 0.01). FACS followed by 16S amplicon sequencing detected differences in bacterial taxa abundance between complement-opsonized, IgA-opsonized and non-opsonized fractions. Functional in-silico profiling revealed associations of complement-opsonization with bacterial virulence factors. Conclusion Luminal and mucosal complement expression and activity is evident in patients with IBD. Complement opsonisation profiles are distinct from IgA opsonisation and show features of specificity regarding recognition of microbial features.

Metabolites Composition of Bacillus subtilis HussainT-AMU Determined by LC-MS and Their Effect on Fusarium Dry Rot of Potato Seed Tuber

Fusarium dry rot is considered to be the most critical soilborne and postharvest disease that damages potato tubers worldwide when they are stored for a long time. This study was performed to demonstrate the effect of crude extract, culture filtrate, and cell suspension obtained from the bacterium Bacillus subtilis HussainT-AMU, on the net house and field. From oil-contaminated soil, through the serial dilution method, biosurfactant bacteria were isolated on nutrient agar medium. To isolate and screen the prospective biosurfactant strain, various biosurfactant screening methods were used. Standard protocols were carried out for morphological, molecular, and chemical characterization. The disease incidences were significantly decreased under net house and field conditions after treatment with biosurfactant extract (20%, 70%), culture filtrate (23%, 49%), and bacterial cell suspension (46%, 27%), respectively. These results indicate a significant effect of B. subtilis HussainT-AMU against Fusarium oxysporum. The characterization of the partially purified extract by gas-chromatography indicated the existence of a high amount of methyl ester fatty acids. The quantitative analysis determined the presence of lipopeptide-surfactin, fengycin, and iturin in the biosurfactant extract. Scanning electron microscopy revealed that morphological variation was revealed in the structure of stomata size in leaves and in the mycelia of the pathogen that was investigated. This promising biocontrol agent can be a plausible option for further research and can also be used for the production of green biomolecules at an additional industrial level.

Diketopyrrolopyrrole Derivatives as Photosensitizing Agents against Staphylococcus aureus.

Diketopyrrolopyrrole (DPP) derivatives containing sulfonamide (Sulfonamide-DPP), pyridyl (Dipyridyl-DPP) and N-methylpyridyl (MePyridyl-DPP) substituents were assessed as antibacterial photosensitizers. Non-charged DPPs showed an intense absorption band centered at about 480 nm and green fluorescence emission (ΦF ~ 0.7) in acetonitrile. The absorption of MePyridyl-DPP was bathochromically shifted at 510 nm, with decreased fluorescence emission. Sulfonamide-DPP and Dipyridyl-DPP photosensitized the formation of O2 (1 Δg ) (ΦΔ ~ 0.15-0.17), while the production induced by MePyridyl-DPP was at least 10 times lower. Furthermore, these DPPs produced a photoreduction of NBT similar to that of the control. Photodynamic inactivation induced by DPPs was first investigated at the single-bacterium level of Staphylococcus aureus attached to a surface. After 30 min irradiation, MePyridyl-DPP produced a complete eradication of the bacteria. In bacterial cell suspensions, dicationic DPP induced more than 7 log10 decrease in S. aureus cell survival after 30 min irradiation. Potentiation with iodide anions allowed a complete elimination of bacteria after 15 min therapy. This compound was also effective to eliminate S. aureus cells on biofilms. The results show that MePyridyl-DPP bearing two positive groups provides an amphiphilic character to the structure that improves the interaction with the cell envelop. This effect enhances the photocytotoxic activity of MePyridyl-DPP against bacteria.

Antagonistic potential of an Egyptian entomopathogenic nematode, compost and two native endophytic bacteria isolates against the root-knot nematode (Meloidogyne incognita) infecting potato under field conditions

BackgroundThe root-knot nematode, Meloidogyne spp., are one of the most dominant and dangerous group of pests. The deformations and discolorations make tubers unmarketable and/or of less quality. Therefore, management of Meloidogyne spp. becomes an obligatory challenge that warrants intervention. Biological control agents are the best alternative tools for controlling plant-parasitic nematodes that comply with the requirements of the development of the green agriculture and that reduce the reliance on these harmful chemicals. Therefore, this study aimed to evaluate the effectiveness of compost singly, and in combinations with the bio-agents Heterorhabditis bacteriophora, and two bacterial isolates Nem 212 and Nem 213 against the root-knot nematode Meloidogyne incognita infecting potato plants under field conditions.ResultsAmong 15 bacterial isolates (Nem205-Nem219) obtained from the rhizosphere of tomato and eggplant from Giza, Egypt, the two isolates (Nem 212 and Nem 213) were molecularly characterized based on the partial 16S rDNA sequencing analysis. These two bacterial isolates were deposited in the GenBank as Bacillus cereus Nem 212 and B. cereus Nem 213 and were tested against M. incognita J2s in vitro. Results showed that the cell filtrates of B. cereus Nem 212 and B. cereus Nem 213 gave the highest percentage of M. incognita J2s mortality (100%), after 48 h of the in vitro application. Moreover, all the applied treatments significantly suppressed the reproductive of M. incognita on potato plants and enhanced the potato crop yield under the field conditions. Compost enriched with B. cereus Nem 212 cell suspension was the most effective treatment. The combination between the bacterial cell suspension and the compost offered an increase in the disease curing and the potato plant growth and yield production, compared to the treatment with compost alone. The entomopathogenic nematode, Heterorhabditis bacteriophora, was relatively less effective in controlling M. incognita on potato, compared to B. cereus Nem 212 and/or B. cereus Nem 213 treatments. However, when compost was enriched with H. bacteriophora, it increased its capability to control the nematodes.ConclusionsThis study provides insights into the practical usage of EPNs H. bacteriophora, and the endophytic bacteria (B. cereus Nem 212 or B. cereus Nem 213) as biocontrol agents against M. incognita on potato plants. The application of compost enriched with the bacterial cell suspensions of either B. cereus Nem 212 or B. cereus Nem 213 and H. bacteriophora within Galleria mellonella cadaver proved efficient control of M. incognita infecting potato plants and improved the growth and yield of potato plants under field conditions.

Management of Bacterial Blight of Clusterbean Caused by Xanthomonas axonopodis pv. cyamopsidis under Field Conditions

Background: Bacterial blight pathogen (Xanthomonas axonopodis pv. cyamopsidis) of clusterbean causes considerable damage every year during Kharif season and sometimes become very severe, which results in heavy loss in grain yield. Methods: On the basis of in vitro studies, most effective botanical, antibiotic and fungicide at highest concentration were used as individual and its combinations in field experiment during two consecutive years Kharif 2018 and 2019. Seeds of clusterbean cultivar RGC-936 were artificially inoculated with X. axonopodis pv. cyamopsidis by soaking in bacterial cell suspension (2.5x108 cfu/ml) for 30 min and dried under shade. The artificially inoculated seeds were used for sowing in field trial Result: Field evaluation, the combined treatment, seed treatment with streptocycline @ 250 ppm followed two foliar sprays of streptocycline @ 250 ppm and 1st foliar spray of copper oxychloride @ 0.3 per cent and 2nd foliar spray of streptocycline @ 250 ppm at 15 days interval was found the most effective in reducing the per cent disease severity and increasing the grain and fodder yield in clusterbean.

Removal of Fluoride from Synthetic Wastewater Using Carbonised Saw Dust and Suspended and Immobilised Culture of Pseudomonas oleovorans Strain NITD 20 – A Comparative Study

In the present research, three agents such as saw dust (carbonised), waste of the timber industry, suspended bacterial cells of Pseudomonas oleovorans strain NITD 20 and immobilised whole live bacterial cells were used individually for fluoride removal from synthetic wastewater. Carbonised saw dust was used first as an adsorbent at optimum conditions such as pH 7±0.2, initial fluoride concentration of 15 mg·L-1, particle size of 0.10 mm, adsorbent dose of 60 g·L-1, contact time of 10 h and stirring speed of 120 rpm, and it showed 79.04±0.196% fluoride removal. In the present study, both suspended and immobilised cells of ‘Pseudomonas oleovorans strain NITD 20’ were used for the defluoridation process. Immobilisation was done onto the carbonised saw dust. The maximum removal was observed for suspended cells at 94.5±2.1% and immobilised cells at 98±1.23% in 10 h and 8 h incubation periods, respectively, from 15 mg·L-1 fluoride containing synthetic wastewater.