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Isolation and identification of infected sour orange roots revealed the recovery of seven Phytophthora spp. Isolates from seven different locations. On Potato Dextrose Agar (PDA), the fungal colonies were characterized by a white to creamy coloration with a cottony or fluffy appearance, rapid growth, and irregular margins. Pathogenicity tests demonstrated that all isolates caused a significant reduction in the germination percentage of radish seeds grown on Water Agar medium. However, the isolates varied considerably in their effects on seed germination compared to the control treatment, which showed 100% germination, whereas the values ranged between 0% and 36%. Furthermore, the bacterium Lysinibacillus sphaericus, employed as a biocontrol agent against the pathogenic fungus Phytophthora nicotianae, was confirmed by PCR with specific forward and reverse primers, showing approximately 90% similarity to the identified Italian isolate (accession number HM462440.1). Antagonistic activity assays demonstrated the ability of L. sphaericus at a concentration of (160 × 105 CFU) on PDA medium to completely inhibit the mycelial growth of P. nicotianae, where the mean radial growth was 0.00 cm compared to 9.00 cm in the control treatment, corresponding to 100% inhibition relative to 0.00% in the control. In addition, the results indicated positive detection of L. sphaericus production of chitinase and protease enzymes.

The current study aims to exploit the zero-cost inducer wheat bran (WB), an agro-industrial byproduct, for production of alkaline protease (ALK-PR23) by the hyper producer psychrotolerant Lysinibacillus sphaericus Strain AA6 EMCCN3080 for the first time ever. Incubation temperature (25 °C), yeast extract concentration, agitation speed (150 rpm), and aeration ratio [1 volume (liquid):5 volume (Erlenmeyer flask)] provoked ALK-PR23 production; OVAT inferences. The pH, yeast extract, and (NH 4 ) 2 SO 4 levels substantively triggered ALK-PR23 production as deduced from Plackett–Burman design. Incubation time (3 days) and WB [2% (w/v)] were the optimal values inducing positive significant influence on ALK-PR23 as conferred from steepest ascent experiments. Yeast extract (0.446% w/v), (NH 4 ) 2 SO 4 (0.339% w/v), and pH (6.872) prompted ALK-PR23 (592.5 U/mL) with an impressive 98-fold enhancement; Box-Behnken design and ridge steepest ascent path implications. The laboratory validation of the model achieved 100% of the predicted value. Laboratory data would present an eco-friendly, cheap, efficient approach towards concurrent WB recycling and massive production of alkaline protease (ALK-PR23) from L. sphaericus Strain AA6 EMCCN3080 . Present data would greatly encourage unveiling biochemical characteristics of ALK-PR23 in prospective studies. Graphical Abstract

The aim of the study was to characterization of polyhydroxybutyrate (PHB) produced by novel bacterium Lysinibacillus sphaericus BBKGBS6 isolated from soil. The present study reports that the strain L. sphaericus BBKGBS6, which was isolated from agricultural soil and is capable of producing PHB. Extraction of PHB was done by solvent extraction method. The results indicated the presence of crotonic acid and confirmed the presence of polyhydroxybutyrate in the sample. The FTIR spectra were observed characteristic absorption bonds for ester and the presence of C=O and C–O were obtained. GCMS results showed the major molecular fragmentation were, 117 m/z (C5H9O3+), 104 m/z (C4H7O3+), 74 m/z (C3H6O2+), 61 m/z (C2H3O2+), 43 m/z (C2H3O), 59 m/z (C2H3O). 1H and 13C NMR spectra were recorded using purified samples. The molecular weight of PHB (5.64 × 105) was estimated based on viscosity measurement. Films were prepared by the solvent casting method. The structure of crystalline polymers can be determined or refined through best fitting of X-ray powder diffraction profiles. The procedures provide results grossly in agreement with respect to the confirmation of the chain in the powder but differing significantly on a more detailed scale. Such differences represent an additional reason of interest in performing a new structural study. Differential scanning calorimetric experiments was performed using Universal V4.5A TA Instruments, (m.p. 156.61 °C; ΔH = 28.54 J/g) USIC Dharwad. The crystallinity (Xc) of PHB is calculated as per equation given below Xc=DHf×100/DH0×W. The PHB extracted, PHB Sigma and PHB–TS showed two endothermal peaks in between 140 and 200 °C. The enthalpy of melting (ΔHf) was 28.09 J/g for standard PHB and for extracted one is 56.42 J/g. The glass transition temperature of the sample was 140 °C and amorphous temperature was 176.08 °C. Thermo gravimetric analysis (TGA) is a method of thermal analysis in which changes in physical and chemical properties of materials are measured as a function of increasing temperature (with constant heating rate), or as a function of time (with constant temperature and/or constant mass loss). The decomposition temperature at a 10% level determined by TGA for pure PHB in ScCO2 at 70 °C and 22 MPa was 293.32 °C. Tensile strength of PHB film (28.23 Mpa) was carried out according to ASTMD 882 using universal testing machine (Model Lx 5, LYOD ISNT). Water vapor transmission rate of PHB film (29 g/m2/day) was measured as per ASTM E96-95 and carried out according to the desiccant method. Oxygen transmission rate of PHB film 472.36 (cc/m2/day/atm 65% Rh and 27 °C) was measured as per the method of ASTM D-1434-66.

Larvicides based on the bacteria Bacillus thuringiensis svar. israelensis (Bti) and Lysinibacillus sphaericus are effective and environmentally safe compounds for the control of dipteran insects of medical importance. They produce crystals that display specific and potent insecticidal activity against larvae. Bti crystals are composed of multiple protoxins: three from the three-domain Cry type family, which bind to different cell receptors in the midgut, and one cytolytic (Cyt1Aa) protoxin that can insert itself into the cell membrane and act as surrogate receptor of the Cry toxins. Together, those toxins display a complex mode of action that shows a low risk of resistance selection. L. sphaericus crystals contain one major binary toxin that display an outstanding persistence in field conditions, which is superior to Bti. However, the action of the Bin toxin based on its interaction with a single receptor is vulnerable for resistance selection in insects. In this review we present the most recent data on the mode of action and synergism of these toxins, resistance issues, and examples of their use worldwide. Data reported in recent years improved our understanding of the mechanism of action of these toxins, showed that their combined use can enhance their activity and counteract resistance, and reinforced their relevance for mosquito control programs in the future years.

Excessive salinity reduces crop production and negatively impacts agriculture worldwide. We previously isolated endophytic bacterial strains from two halophytic species: Artemisia princeps and Chenopodium ficifolium . We used three bacterial isolates: ART-1 ( Lysinibacillus fusiformis ), ART-10 ( Lysinibacillus sphaericus ), and CAL-8 ( Brevibacterium pityocampae ) to alleviate the impact of salinity stress on rice. The impact of 160 mM NaCl salinity on rice was significantly mitigated following inoculation with these bacterial strains, resulting in increased growth and chlorophyll content. Furthermore, OsNHX1 , OsAPX1 , OsPIN1 and OsCATA expression was increased, but OsSOS expression was decreased. Inductively coupled plasma mass spectrometry (ICP-MS) revealed reduced K + and Na + levels in shoots of bacteria-inoculated plants, whereas that of Mg 2+ was increased. Bacterial inoculation reduced the content of total flavonoids in rice leaves. Salinized plants inoculated with bacteria showed reduced levels of endogenous salicylic acid (SA) and abscisic acid (ABA) but increased levels of jasmonic acid (JA). In conclusion, the bacterial isolates ART-1, ART-10, and CAL-8 alleviated the adverse effect of salinity on rice growth, which justifies their use as an eco-friendly agricultural practice.

Low temperature is an important abiotic variable that inhibits plant growth and yield by restricting plant distribution on land. Cold-tolerant plant growth-promoting rhizobacteria (PGPR) improve nutrient absorption and availability in plants through biochemical and physiological mechanisms. Furthermore, they increase the tolerance of plants to cold stress. Different strains of bacteria were isolated from the roots of Suaeda nudiflora. These isolates were identified using 16SrDNA as Lysinibacillus fusiformis strain YJ4 and Lysinibacillus sphaericus strain YJ5 and were used to study their role in alleviating the harmful effect of cold stress. The two bacterial strains have the ability to solubilize phosphorus and to produce gluconic acid, phytohormones, catechol and hydroxymate siderophores. The present study aimed to study the effect of inoculating maize seeds with PGPR and its use to alleviate the adverse effects of cold stress. The results showed that cold stress (4 °C) reduces germination, growth criteria, photosynthetic pigments (i.e., chl a, chl b, and carotenoids), photosynthetic rate, membrane stability index, phytohormones (auxin and gibberellin), and mineral contents (N, P, K, and Ca) while increasing conductivity, malondialdehyde (MDA), lignin, cell viability, osmolytes (proline, glycine betaine, and soluble sugars), phenolic content, abscisic acid, 1‑aminocyclopropane-1-carboxylic acid (ACC) content and the antioxidant defense system in maize plants. Besides, the lignification, osmolytes, phenolic content, phytohormones, the enzymatic antioxidant defenses (i.e., superoxide dismutase, catalase, and phenylalanine ammonia-lyase), and mineral contents of maize plants increased after inoculation with L. fusiformis and L. sphaericus alone or in combination as compared to normal and cold stress conditions. In conclusion, the inoculation with L. fusiformis and L. sphaericus in maize plants induced resistance of osmotic and oxidative stress caused due to exposure to cold stress by upregulation of osmolytes, phenolics, phytohormones, and antioxidant enzymes. Also, L. sphaericus strains is more effective in tolerance to cold stress than L. fusiformis.

S-methoprene, an insect growth regulator, and Lysinibacillus sphaericus ( Ls ), an entomopathogenic bacterium, are important larvicides used to control Culex pipiens [L.] mosquitoes, the primary vector of West Nile virus, in the Chicago, IL USA region. Resistance to both agents has been documented globally including a report of resistance ratios greater than 100 to S-methoprene in the Chicago region. Laboratory studies have suggested the potential for unidirectional cross-resistance between S-methoprene and Ls , despite differing modes of action. Among wild populations of Cx. pipiens in the Chicago area, this study aimed 1) to assess resistance status to Ls , 2) confirm the presence of S-methoprene resistance ratios >100, 3) determine if higher S-methoprene resistance ratios are associated with higher Ls resistance ratios, or whether Ls resistance arises solely from Ls exposure, and (4) determine the relationship between Ls treatment history and resistance levels of that active ingredient. We assessed susceptibility to both S-methoprene and Ls in 32 Cx. pipiens populations: 19 with S-methoprene exposure but no Ls history, and 13 with multi-year exposure to both larvicide active ingredients. Ls susceptibility was evaluated using dose-response bioassays to estimate LC 50 , LC 90 , and resistance ratios. Susceptibility to S-methoprene was tested using diagnostic doses corresponding to resistance ratios of 10 and 100 at the LC 50 . Resistance ratios to S-methoprene exceeding 10 were detected in 30 of 32 sampled populations. Among the 13 sites with prior Ls exposure, 11 were observed with resistance ratios > 5. In contrast, none of the 19 populations without Ls exposure exhibited Ls resistance, despite exhibiting higher S-methoprene resistance ratios. This lack of overlap supports the conclusion that S-methoprene resistance does not confer cross-resistance to Ls in the studied region. Logistic regression revealed a strong association between Ls treatment history and resistance development. The probability of Ls resistance exceeded 80% after 10 Ls applications within an eight-year period. These findings emphasize the need to develop improved resistance management strategies for larvicidal insecticides.

An isolate of a Gram-positive, strictly aerobic, motile, rod-shaped, endospore forming bacterium was originally isolated from soil when screening and bioprospecting for plant beneficial microorganisms. Phylogenetic analysis of the 16S rRNA gene sequences indicated that this strain was closely related to Lysinibacillus fusiformis NRRL NRS-350 T (99.7%) and Lysinibacillus sphaericus NRRL B-23268 T (99.2%). In phenotypic characterization, the novel strain was found to grow between 10 and 45 °C and tolerate up to 8% (w/v) NaCl. Furthermore, the strain grew in media with pH 5 to 10 (optimal growth at pH 7.0). The predominant cellular fatty acids were observed to be iso-C 15: 0 (52.3%), anteiso-C 15: 0 (14.8%), C 16:1 ω7C alcohol (11.2%), and C 16: 0 (9.5%). The cell-wall peptidoglycan contained lysine-aspartic acid, the same as congeners. A draft genome was assembled and the DNA G+C content was determined to be 37.1% (mol content). A phylogenomic analysis on the core genome of the new strain and 5 closest type strains of Lysinibacillus revealed this strain formed a distinct monophyletic clade with the nearest neighbor being Lysinibacillus fusiformis . DNA–DNA relatedness studies using in silico DNA–DNA hybridizations (DDH) showed this species was below the species threshold of 70%. Based upon the consensus of phylogenetic and phenotypic analyses, we conclude that this strain represents a novel species within the genus Lysinibacillus , for which the name Lysinibacillus pinottii sp. nov. is proposed, with type strain PB211 T (= NRRL B-65672 T , = CCUG 77181 T ).

Lysinibacillus sphaericus, an endophytic diazotroph from rice (Oryza sativa), was investigated for colonization and growth-promoting effects. Seed bacterization introduced the endophyte, confirmed through re-isolation of antibiotic media, microscopy, and molecular identification. L. sphaericus showed efficient colonization in rice tissues, maintaining stable populations across plant parts. Light and scanning electron microscopy revealed intracellular colonization in roots of inoculated plants. The endophyte enhanced plant growth, nutrient uptake, and nitrogen fixation compared with uninoculated controls and Pseudomonas fluorescens-treated plants. Increased production of phytohormones, including indole-3-acetic acid, gibberellic acid, and cytokinins, was observed in L. sphaericus-inoculated plants, correlating with improved root and shoot development. The endophyte demonstrated biocontrol activity against rice sheath blight pathogen Rhizoctonia solani, with plants showing reduced disease severity. L. sphaericus populations remained stable in host tissues following pathogen challenge. These findings highlight L. sphaericus's potential as an endophytic diazotroph for sustainable rice cultivation, growth promotion, nutrient acquisition, and disease suppression.

Piezophiles, by the commonly accepted definition, grow faster under high hydrostatic pressure (HHP) than under ambient pressure and are believed to exist only in pressurized environments where life has adapted to HHP during evolution. However, recent findings suggest that piezophiles have developed a common adaptation strategy to cope with multiple types of stresses including HHP. These results raise a question on the ecological niches of piezophiles: are piezophiles restricted to habitats with HHP? In this study, we observed that the bacterial strains Sporosarcina psychrophila DSM6497 and Lysinibacillus sphaericus LMG 22257, which were isolated from surface environments and then transferred under ambient pressure for half a century, possess moderately piezophilic characteristics with optimal growth pressures of 7 and 20 MPa, respectively. Their tolerance to HHP was further enhanced by MgCl₂ supplementation under the highest tested pressure of 50 MPa. Transcriptomic analysis was performed to compare gene expression with and without MgCl₂ supplementation under 50 MPa for S. psychrophila DSM6497. Among 4390 genes or transcripts obtained, 915 differentially expressed genes (DEGs) were identified. These DEGs are primarily associated with the antioxidant defense system, intracellular compatible solute accumulation, and membrane lipid biosynthesis, which have been reported to be essential for cells to cope with HHP. These findings indicate no in situ pressure barrier for piezophile isolation, and cells may adopt a common adaptation strategy to cope with different stresses.