Fusarium stem rot disease caused by Fusarium verticillioides has become one of the most serious issues confronting Indonesian farmers in recent years. An alternative option for suppressing this disease is to use indigenous microbes as an eco-friendly method to reduce synthetic fungicides. The objective of the research was to identify the molecular characteristics and effectiveness of an indigenous microbial consortium in controlling Fusarium stem rot disease. Identification of indigenous microbes is carried out based on molecular characters using universal primers, namely ITS-1/ITS-4 for fungi and 27F/1492R for bacteria. Nucleotide sequences were analyzed using Bioedit 7.2 version and MEGAX software. In vitro testing was carried out using the dual culture method for indigenous fungi and the disc diffusion method for indigenous bacteria. Meanwhile, in planta testing was conducted by evaluating a consortium of fungi and bacteria to control F. verticillioides in the field using a randomized block design with three replications, followed by a 5% DMRT test. The use of universal primer pairs ITS-1/ITS-4 and 27F/1492R succeeded in amplifying DNA bands of indigenous microbial isolates measuring ±600 bp and ±1465 bp, respectively. S6 and S9 bacterial isolates were identified as Bacillus cereus. JRP 7 and SEDF 6A isolates were identified as Trichoderma asperellum and JRP 10 isolate was identified as Penicillium raperi. All identification resulted in homology of >99%. The in vitro inhibitory reactions of isolates JRP 7, JRP 10, SEDF 6A, S6, and S9 against F. verticillioides were >60%. Disease severity of B6B9C10, B6B9C6, B6B9C7, B9C6C7, B9C7C10, and C6C7C10 treatments significantly proved their ability to control F. verticillioides in the field with a lower percentage of disease severity than positive controls, which are 23.33%, 18.89%, 23.33%., 21.85%, 14.07%, and 15.93%, respectively. The B9C7C10 consortium (S9 + JRP 7 + JRP 10 isolates) containing three species of indigenous microbes, i.e. B. cereus, T. asperellum, and P. raperi is the most effective at controlling F. verticillioides and may be developed for use as biopesticide products. Antagonist; Bacteria; Consortium microbes; Fungi; Molecular. It is pFusarium stem rot disease, caused by Fusarium verticillioides, has recently attacked several Indonesian maize centers  gene and the internal transcribed spacer (ITS) ribosomal DNA (rDNA) region is the most frequently used identification method to identify bacteria and fungi .The 16S rRNA gene is a conserved gene sequence shared by all bacterial species. These gene sequences provide important information for the identification of all species. The 16S rRNA gene is very effective because it has high accuracy and fast identification . While tFusarium stem rot disease.Currently, the use of biological agents in controlling plant pathogens is limited to a single application, making them less effective in the field. Considering the success rate of single fighter application of biological agents that have been used so far is still stagnant and tends to be ineffective, it is necessary to study the model of using multiple fighter-based biological agents or a consortium ofnumerous types of fungi and bacteria thatmay effectively and efficiently suppress plant pathogen attacks. The objective of this research was to examine the molecular characteristics of indigenous microbes and the effectiveness of the indigenous microbial consortium in controlling 22.1The indigenous microbial isolates used in this study included 3 isolates of fungi collected from earlier studies, i.e SEDF 6A MRS, JRP 7 MRS, and JRP 10 MRS , as well2.22.2.1The 7-days-old fungal isolates on PDA media were suspended using sterile distilled water. A total of 100 ml of sterile distilled water was added to the fungal isolates and then the conidia and mycelium were harvested using a spatula. Conidia and mycelium suspension of the indigenous fungi were put in collection bottles. Meanwhile, indigenous bacterial isolates were cultured on nutrient broth (NB) media. One loopful of indigenous bacterial isolate from the stock was put into an Erlenmeyer flask with 100 ml of NB media. The Erlenmeyer flask was then shaken on a rotary shaker at 180 rpm for 48 h at room temperature. The indigenous bacterial suspension was put in a collection bottle. Each suspension of fungi and bacteria was put into a 1.5 ml microtube and then centrifuged at 10,000 rpm for 5 min. The supernatant from the centrifuge was discarded while the precipitate/pellet was used for DNA extraction.2.2.2DNA Extraction of indigenous microbe was carried out using the Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit. The working principle of this Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit is fungal and/or bacterial samples are added directly to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) and rapidly and efficiently lysed by bead beating without using organic denaturants or proteinases. The DNA is then isolated and purified using our Zymo-Spin™ Technology and is ideal for downstream molecular-based applications including PCR, array, etc.2.2.32O, 1 μl of primer forward, 1 μl of primer reverse, and 2 μl of DNA template. The primer used is a universal primer, i.e primer ITS-1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS-4 (5′TCCTCCGCTTATTGATATGC-3′) for fungi and primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) for bacteria. The PCR cycle consists of an initiation or predenaturation step at 95 °C in 3 min which is followed by 35 cycles of the denaturation stage at 95 °C in 15 s, annealing stage at 55 °C in 30 s, extension stage at 72 °C in 1 min, and final extension stage in 3 min at 72 °C. Indigenous microbial DNA was electrophoresed at a voltage of 110 V for 50 min. The results of the electrophoresis were visualized with a UV transilluminator and photos were taken using the camera.DNA amplification was carried out by referring to the method carried out by 2.2.4The amplification product is sent to FirstBase  for sequencing. Sequence results were analyzed using the basic local alignment search tool (BLASTN 2.13.0+) with an optimization program to obtain the sequence of DNA bases contained in the site of National Center for Biotechnology Information (NCBI). The nucleotide sequence results obtained were then analyzed using multiple alignment ClustalW on software Bioedit Sequence Alignment Editor 7.2 version. The kinship relationship between isolates was constructed using the software Molecular Evolutionary Genetic Analysis Software 10.0 version (MEGAX) with bootstrap 1000 times repetitions.2.32.3.1F. verticillioides on PDA media in the same Petri dish. Fusarium verticillioides isolates were cultured 3 days earlier than the indigenous fungi. The position of each mold is arranged facing each other with a distance of 3 cm. Media that was inoculated only by F. verticillioides acted as control. Cultures were incubated at room temperature for 7 days accompanied by the observation of the radial growth of the colonies. Percentage growth inhibition (PGI) is determined based on F. verticillioides which grows against the antagonist microorganism, and R2 is the radius of the F. verticillioides colony that grows towards the antagonist microorganism PGI=D2.4The compatibility of each indigenous microbe isolate in the consortium was tested using the antagonist test . A compa2.52.5.1F. verticillioides was carried out on sterile potato dextrose broth (PDB) media. Pure inoculum of F. verticillioides was propagated from a stock isolate of F. verticillioides which were first cultured on PDA media and incubated for 7 days at room temperature. Thereafter, the conidia were harvested from the 7-day-old culture of F. verticillioides and put into an Erlenmeyer flask containing 100 ml of PDB medium, then incubated for 4 days on a rotary shaker at 180 rpm at room temperature.Multiplication of 2.5.2F. verticillioides on maize crops in the field using Anoman variety maize seeds. Maize seeds were treated with hot water treatment at 55 °C for 20 min. The 7-days-old cultures of fungal isolates and the 48-hours-old bacterial isolates were suspended by adding sterile distilled water to the fungi and bacteria isolates, then scraped with a spatula and then used to soak the maize seeds. Maize seeds were soaked according to the indigenous microbial consortium treatment in Erlenmeyer containing a suspension of indigenous microbe isolates by shaking at 170 rpm for 24 h. The positive control treatment was carried out by soaking maize seeds that had been treated with hot water treatment using sterile distilled water, while the negative control treatment was soaked with a synthetic fungicide using active copper oxide 56%.The seed soaking method was used to test the ability of the indigenous microbial consortium to suppress the attack intensity of 2.5.32.5.3.1The experimental land was set up in a full-tillage system by using a four-wheel tractor. Each treatment has a 3 × 5 m plot with 75 × 20 cm spacing and two seeds per hole. During planting and crop emergence, Furadan 3 G was applied at a rate of 8–10 kg/ha. First fertilization was at 7–10 days after planting (DAP) with 350 kg/ha NPK Phonska (15:15:15) and 150 kg/ha urea. After fertilizing, 25 plants per row were selected. The second fertilization was at 30 DAP with Urea at 250 kg/ha and NPK Phonska at 100 kg/ha.The third fertilization was at 45–50 DAP with Urea at 150 kg/ha, based on the leaf color chart. To ensure plant health, irrigation, weeding, pest and disease control were continuously monitored.2.5.3.26 conidia/mL) and bacteria (concentration 1 × 107−9 colonies/ml) taken 100 ml and put into 700 ml PDB medium, then added with 10 g of molasses as microbial nutrition. The indigenous microbial consortium suspension was then incubated again at room temperature by shaking at 170 rpm for 48 h. Tested seeds were soaked in the microbial consortium based on each treatment at 160 rpm for 24 h. The soaked seeds were planted in the experimental field. Besides, the microbial consortium application was performed by spraying the suspension from the bottom to the top of the maize stem at 21, 28, 35 and 42 DAP. The indigenous microbe consortium treatments are (1) B6B9C6: Isolate S6 + S9 + SEDF 6A MRS + molasses; (2) B6B9C7: Isolate S6 + S9 + JRP 7 MRS + molasses; (3) B6B9C10: Isolate S6 + S9 + JRP 10 MRS + molasses; (4) B6C6C7: Isolate S6 + SEDF 6A MRS + JRP 7 MRS + molasses; (5) B6C6C10: Isolate S6 + SEDF 6A MRS + JRP 10 MRS + molasses; (6) B6C7C10: Isolate S6 + JRP 7 MRS + JRP 10 MRS + molasses; (7) B9C6C7: Isolate S9 + SEDF 6A MRS + JRP 7 MRS + molasses; (8) B9C6C10: Isolate S9 + SEDF 6A MRS + JRP 10 MRS + molasses; (9) B9C7C10: Isolate S9 + JRP 7 MRS + JRP 10 MRS + molasses; (10) C6C7C10: SEDF 6A MRS + JRP 7 MRS + JRP 10 MRS + molasses; (11) Control (-): Synthetic fungicide; (12) Control (+): Distilled water.The fungi isolates, i.e SEDF 6A MRS, JRP 7 MRS, and JRP 10 MRS, were cultured and propagated on PDB media and then incubated at room temperature by shaking at 170 rpm for 120 h. Meanwhile, bacteria isolates, i.e S6 and S9, were cultured and propagated using NB media and incubated at room temperature by shaking at 170 rpm for 48 h. Fungal and bacterial isolates were consorted based on the treatment, using of each suspension of fungi  into the second stem segment. A week following the initial inoculation, the second inoculation was performed by injuring the second segment from the base of the stem with a cutter that had been dipped in suspension. F. verticillioides.The first inoculation of the pathogen 2.5.5Fusarium stem rot disease severity was assessed on 90 DAP plants. Observations were made by taking 10 plant samples and scoring them based on the appearance of infection symptoms using the modified disease scale ]; scale 2 [infection spreads over a quarter of the inoculated internodes length in the pith and cortex tissue (>10–20%)]; scale 3 [infection spreads to half of the inoculated internodes length in the pith and cortex tissue (>20–30%)]; scale 4 [Infection spreads three-quarters of the inoculated internodes length in the pith and cortical tissue (>30–40%)]; scale 5 ; scale 6 [infection increases across the nodal plate and spreads to adjacent segments of the inoculated segment (>50–60%)]; scale 7 ; scale 8 ; and scale 9  . The disease scale is then transformed into the intensity percentage of disease formula as follows I=∑n×2.64, 0.1 ml of the suspension was cultured on PDA medium for fungus and NA medium for bacteria. Multiple subcultures of microorganisms growing on each medium were repeated until a pure culture was produced and was suitable for identification.Soil from the rhizosphere and stem tissue of maize plants that were pre-applied with indigenous microbe were taken and weighed 10 g each. The soil and maize stem were crushed separately using a sterile mortar by adding 10 ml of sterile distilled water. Each Erlenmeyer flask added 10 mL of the suspension, 90 mL of distilled water and was homogenized for 30 min by a vortex. One millilitre of the homogenized suspension was diluted to 102.7F. verticillioides in the field using a randomized block design consisting of 10 consortium treatments, positive and negative control, then replicated three times. The data obtained were then statistically analyzed with analysis of variance and followed by Duncan's Multiple Range Test (DMRT) at the 5% significance level (0.05).Antagonis test of indigenous microbial isolates was arranged using a completely randomized design. The treatments consisted of 5 indigenous microbe isolates, positive and negative control. Each treatment was replicated 3 times. Observational data were statistically analyzed with analysis of variance and followed by Least Significant Difference (LSD) at the 5% significance level (0.05). Meanwhile, the effectiveness of indigenous microbial consortium against 33.1The use of universal primer pairs ITS-1/ITS-4 and 27F/1492R successfully amplified DNA bands of indigenous microbe isolates measuring ±600 bp and ±1465 bp, respectively . The ITSTrichoderma asperellum strain unilaGT from Lampung-Indonesia (99.7%), T. asperellumisolate WT13 from India (100%), T. asperellum strain MLT5J1 from Yogyakarta-Indonesia (100%), T. asperellum strain ACCC32915 from China (100%), and T. asperellum strain UC1 (MF774876) from Malaysia (99.7%). While the JRP 10 isolate have similarities to Penicillium raperi strain CMV006I1 from South Africa (100%), P. raperi strain DTO 249-D2 from Netherlands (100%), P. raperi strain IA32 from India (100%), and P. raperi clone SF_761 from China (100%). For the level of similarity between the sample isolates, JRP 7 and SEDF 6A, the similarity level was 100%, while the JRP 10 isolate had a similarity level of 72.70% to JRP 7 and SEDF 6A isolates.The results of DNA sequence analysis of JRP 7, JRP 10, and SEDF 6A isolates based on ITS regional amplification revealed distinct identities for each of these fungal isolates with a similarity level of >99% . The resBacillus cereus strain WHX1 from China (99.9%), B. cereus strain YM16 from Malaysia (100%), B. cereus strain ABCFI from India (100%), B. cereus strain 156gite from France (99.8%), B. cereus strain NK1 from Japan (99.8%), B. cereus strain FORT 154 from Brazil (100%), B. cereusstrain 44 from Argentina (100%), and B. cereus strain CZW-9 from China (100%). Meanwhile, S9 isolate had similiarities to Bacillus cereus strain WHX1 from China (99.8%), B. cereus strain YM16 from Malaysia (99.7%), B. cereus strain ABCFI from India (99.7%), B. cereus strain 156gite from France (99.9%), B. cereus strain NK1 from Japan (99.9%), B. cereus strain FORT 154 from Brazil (99.7%), B. cereus strain 44 from Argentina (99.7%), and B. cereus strain CZW-9 from China (99.7%). The level of similarity between S6 and S9 isolates was very high, 99.7%.The DNA nucleotide sequences of bacterial isolates based on the amplification of the gene encoding 16S revealed a similarity level of >99% between the S6 and S9 isolates and the genebank isolates in the NCBI . The S6 T. asperellum isolate WT13 from India, strain ACCC32915 from China, and strain MLT5J1 from Yogyakarta-Indonesia with a genetic distance coefficient of 0.000. In addition, JRP 7 and SEDF 6A isolates also showed close kinship. which is close to T. asperellum strain unilaGT from Lampung-Indonesia and strain UC1 from Malaysia with a genetic distance coefficient of 0.002. While JRP 10 isolates had a very close relationship to P. raperi strain CMV006I1 from South Africa, strain DTO 249-D2 from Netherlands, strain IA32 from India, and clone SF_761 from China with a genetic distance coefficient of 0.000. The JRP 7 dan SEDF 6A isolates had a fairly distant kinship to the JRP 10 isolate which is indicated by the coefficient of genetic distance that is far from 0.122 to 0.149  had a similar potential as negative controls using chemical-synthetic fungicides (Nordox® 56WP) to suppress the development of Fusarium stem rot disease in the observation field. Statistical analysis showed that several microbe consortium treatments were significantly different from the positive control (distilled water) to suppress Fusarium stem rot disease at 5% DMRT test. Disease severity of B6B9C10, B6B9C6, B6B9C7, B9C6C7, B9C7C10, dan C6C7C10 treatments were 23.33%, 18.89%, 23.33%, 21.85%, 14.07%, dan 15.93%, respectively. It was showed significantly that the disease severity was lower than the positive control, 30.74% . Isolate JRP 7 had greenishwhite colony color that turns dark green when approaching old age, hyaline hyphae with a septum, and pyramidal conidiophore with the phialide branches, the phialide size, conidia globose-shaped and green, and growth rate of 34.43 mm/day . The characteristics of isolate JRP 7 were similar to T. asperellum characterized by T. asperellum had conidiophores to which branched phialide, 2–3 in number, measuring 6.4 × 1.4 μm. Conidia were colored greenish, ellipsoid, 3.1 × 2.6 μm, and chlamydospores unicellular, terminal, and roughly spherical measuring 20.5 × 21.2 μm. The colony of Penicillium sp. has a cotton-like surface texture with radial grooves and yellowish-white color, and reached maximum growth on day 9th on PDA media. The morphological characteristic of Penicillium sp. was a single-celled chain of conidia produced from specialized conidia cells called phyalids . These characteristics are comparable to those reported by B. cereus on NA culture media showed that the morphology of B. cereus colonies was milky white and irregular shaped with a convex and slightly shiny surface. B. cereus has short rod-shaped cells to single rods with a single arrangement . B. cereus bacteria, i.e white, circular shape, short bacilli with all edges, and convex elevation.Three indigenous microbes that were successfully re-isolated from the tested plants were then identified visually under a microscope. The microbes were identified as their original microbial isolates, i.e. 4T. asperellum with a homology level of >99% and a genetic distance coefficient of 0.000–0.0001. Meanwhile, JRP 10 isolate was identified as P. raperi with 100% homology and a very close genetic distance coefficient of 0.000. Fungal DNA amplification by PCR technique using ITS-1/ITS-4 primer pair amplifies the ITS rDNA region. The ITS region is the genomic coding region for the rDNA components. In addition, this ITS region also has a high probability of identifying the type of fungus because it has the clearest barcode differences between species or there are inter- and intra-specific DNA variations  suppressed the density disease of Fusarium stem rot with the lowest attack percentage of 14.07% by 5% DMRT test. This value indicated a significantly lower disease severity compared to the positive control (30.74%). In the positive control treatment, Fusarium stem rot was seen much more than in the microbial consortium treatment with symptom characteristics, that the lower stem was yellowish and in severe attacks it turned fawn-colored. In addition, symptomatic plants also showed gray and drooping leaves  with indigenous microbes of B. cereus, T. asperellum, and P. raperi was most effective against F. verticillioides that the lowest percentage of disease severity is 14.07%. Considering the field success of multiple fighter-based biological agents, this microbial consortium can be further developed as a biopesticide formulation.Bacterial isolates of S6 and S9 were identified All authors listed have significantly contributed to the development and the writing of this article.Muhammad Aqil and research members were supported by The National Research and Innovation Agency of the Republic of Indonesia through the funding research support on the National Research Priority Grant Programme (PRN 2021).Data included in article/supp. material/referenced in article.The authors declare no conflict of interest.Supplementary content related to this article has been published online at [URL].


We evaluated the in vitro activity of ivermectin on Vero E6 cells infected with 30 clinically isolated SARS-CoV-2 strains belonging to 14 different variants, and particularly 17 strains belonging to six variants of concern (VOC) . The in vitro activity of ivermectin was compared to those of chloroquine and remdesivir. Unlike chloroquine (EC50 from 4.3 ± 2.5 to 29.3 ± 5.2 µM) or remdesivir (EC50 from 0.4 ± 0.3 to 25.2 ± 9.4 µM), ivermectin showed a relatively homogeneous in vitro activity against SARS-CoV-2 regardless of the strains or variants (EC50 from 5.1 ± 0.5 to 6.7 ± 0.4 µM), except for one omicron strain (EC50 = 1.3 ± 0.5 µM). Ivermectin  was, overall, more potent in vitro than chloroquine  (p = 1.3 × 10−34) and remdesivir  (p = 1.6 × 10−13). These results should be interpreted with caution regarding the potential use of ivermectin in SARS-CoV-2-infected patients: it is difficult to translate in vitro study results into actual clinical treatment in patients.Over the past two years, several variants of SARS-CoV-2 have emerged and spread all over the world. However, infectivity, clinical severity, re-infection, virulence, transmissibility, vaccine responses and escape, and epidemiological aspects have differed between SARS-CoV-2 variants. Currently, very few treatments are recommended against SARS-CoV-2. Identification of effective drugs among repurposing FDA-approved drugs is a rapid, efficient and low-cost strategy against SARS-CoV-2. One of those drugs is ivermectin. Ivermectin is an antihelminthic agent that previously showed in vitro effects against a SARS-CoV-2 isolate  with an IC In December 2019, a new disease called coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), began to spread all over the world . Only va50) of around 2.0 µM [One of those drugs is ivermectin. It is an antihelminthic agent that previously showed in vitro effects on RNA and DNA viruses such as Zika virus, dengue virus, West Nile virus, Chikungunya virus and equine herpesvirus type I . Ivermecd 2.0 µM .However, infectivity, clinical severity, re-infection, virulence, transmissibility, vaccine responses and escape, and epidemiological aspects have differed according to the SARS-CoV-2 variants ,35,36,3750 means of ivermectin for the 30 clinically isolated SARS-CoV-2 strains ranged from 1.3 ± 0.5 to 6.7 ± 0.4 µM  . There was a significant difference between the variants analysed  (The EC± 0.4 µM . There wum test) . Only thum test) .50 means of chloroquine, used for comparison, for the 30 clinically isolated SARS-CoV-2 strains ranged from 4.3 ± 2.5 to 33.7 ± 9.0 µM  . The alpha and omicron variants were the most susceptible  (p = 0.69).There was no significant difference in chloroquine activity within a variant  regardless of the strains tested (um test) . Howevervariant) . There w50 means of remdesivir, used for comparison, for the 30 clinically isolated SARS-CoV-2 strains ranged from 0.4 ± 0.3 to 25.9 ± 7.4 µM  . There was a significant difference between the different variants analysed . The omicron variant was the most susceptible to remdesivir  followed by the alpha variant  and by the variant related to Wuhan   regardless of the strains tested (um test) . Only thvariant) .p = 0.0002), these were relatively homogeneous (from 5.1 ± 0.5 to 6.7 ± 0.4 µM) if the susceptibility of the IHU-MI-5227 omicron strain is removed. Indeed, this strain presented a higher susceptibility to ivermectin than the other 29 strains (1.3 ± 0.5 µM). These results were consistent with the previous data by Caly et al. (IC50 = 2.8 µM) [50 = 219, mean EC50 = 5.7 ± 1.0 µM) was more potent in vitro overall than chloroquine   and remdesivir  .Although the 14 SARS-CoV-2 variants showed a significant variation of in vitro susceptibility to ivermectin  . IvermecSeveral modes of action were suggested for ivermectin . Host prUnlike ivermectin, which showed relatively homogeneous in vitro activity against SARS-CoV-2 regardless of the strain or variant, variant susceptibility to chloroquine was heterogeneous. Omicron and alpha variants were the most susceptible to chloroquine. The strains related to Wuhan were less susceptible than strains belonging to the omicron and alpha variants but were more susceptible than strains belonging to the beta, gamma and delta variants. Chloroquine can inhibit in silico viral entry into the host cell by interacting with sialic acids linked to gangliosides on host cellular surface and ACE receptor ,61,62,63The emergence of mutations in the spike glycoprotein of SARS-CoV-2 might impact drug efficacy, and more particularly chloroquine efficacy. As compared to the spike protein relative to the Wuhan sequences, the different variants include few non-synonymous mutations except the omicron variant which contains 30 mutations. The different mutations for each variant are reported in In vitro susceptibility to remdesivir also varied. The three omicron, alpha and Wuhan-related variants were the most susceptible variants. In vitro remdesivir shows a broad spectrum of antiviral activity against RNA viruses by targeting replicase such as RdRp ,72. Remd50 of around 2.0 µM [50 [However, these results must be interpreted with caution regarding the potential use of ivermectin in SARS-CoV-2-infected patients: it is difficult to translate in vitro study results into actual clinical treatment in patients. First, it is crucial to determine whether the concentrations required are consistent with concentrations observed in humans. Ninety-three percent of ivermectin is bound to plasma proteins and there is no data on penetration and concentration of ivermectin into human lungs . Modellid 2.0 µM . Anotherd 2.0 µM . Moreove0 µM [50 .Ivermectin antiviral activity can be improved by combinations with antiviral agents with differing modes of action and new pharmaceutical formulations that can more efficiently deliver ivermectin at high concentrations in the lung tissue. The in vitro combination of ivermectin (2 µM) and remdesivir (6 µM) shows highly synergistic effects against the murine hepatitis virus (MHV), which belongs to the betacoronavirus genus like the SARS-CoV-2 . IvermecThe administration of ivermectin by inhalation could be a way to achieve its accumulation into the lungs. The administration of nebulised ivermectin at 116.5 mg/kg in rats led to plasma concentrations of 186.7 ng/mL (0.21 µM) 24h after administration and 524.3 ng/g in lung tissue 168 h after administration . In piglThe clinical efficacy of ivermectin in COVID-19 treatments remains controversial. In one retrospective study, a single standard dose of 200 µg/kg of ivermectin did not significantly reduce the duration of the SARS-CoV-2 detection and did not improve clinical outcomes in severe COVID-19 patients . HoweverIvermectin and chloroquine diphosphate were bought from Sigma Aldrich  and remdesivir from Apollo Scientific . Stock solutions of ivermectin and remdesivir were prepared in DMSO/water 10% and chloroquine in water. All the stock solutions were then diluted in Minimum Essential Media  in order to have seven final concentrations ranging from 0.1 µM to 100 µM. Final concentrations of DMSO in the assay were under 0.2% and had no influence in viral replication into Vero E6 cells.Thirty clinically isolated SARS-CoV-2 strains were used: five strains closely related to the initial Wuhan isolate  (B lineage) were collected from hospitalised patients during the first COVID-19 outbreak in March–May 2020 in Marseille , four st® CRL-1586™) in MEM with 4% foetal bovine serum and 1% glutamine (complete medium). Vero E6 cells are one of the most widely used cells for the culture of SARS-CoV-2 due to the presence of the high expression of angiotensin converting enzyme 2 (ACE2) receptors, essential for SARS-CoV-2 cell entry [The strains were maintained in production in Vero E6 cells , as previously described [drug concentration − mean CTcontrol 0%)/(mean CTcontrol 100% − mean CTcontrol 0%) × 100. The CTcontrol 0% (0% of inhibition) corresponds to the mean of 12 CT of SARS-CoV-2 replication in the absence of drug 48 h after infection. The CTcontrol 100% (100% of inhibition) corresponds to the mean of 12 CT of SARS-CoV-2 after 48 h of Vero E6 cells infection in the presence of high concentrations of drug. This CTcontrol 0% is similar to the CT of virus inoculum used to infect Vero E6 cells at 0 h.Briefly, 96-well plates were prepared with 5 × 10escribed . The per50 (median effective concentration) values were estimated through nonlinear regression using the R software (ICEstimator version 1.2). EC50 values resulted in the mean of 5 to 11 independent experiments.ECThe in vitro evaluation of ivermectin has been described in only one paper using only one SARS-CoV-2 isolate  . In the


Secondly, we specified αV integrin membrane localization in acrosome-intact and acrosome-reacted sperm and compared its pattern between mouse, pig, and human. Using immunodetection and structured illumination microscopy (SIM), the αV integrin localization was confined to the plasma membrane covering the acrosomal cap area and also to the inner acrosomal membrane of acrosome-intact sperm of all selected species. During the acrosome reaction, which was induced on capacitated sperm, the αV integrin relocated and was detected over the whole sperm head. Knowledge of the integrin pattern in mature sperm prepares the ground for further investigation into the pathologies and related fertility issues in human medicine and veterinary science.Integrins are transmembrane receptors that facilitate cell adhesion and cell–extracellular matrix communication. They are involved in the sperm maturation including capacitation and gamete interaction, resulting in successful fertilization. αV integrin belongs to the integrin glycoprotein superfamily, and it is indispensable for physiological spermiogenesis and testosterone production. We targeted the gene and protein expression of the αV integrin subunit and described its membrane localization in sperm. Firstly, in mouse, we traced  Integrin αV  belongs to the heterodimeric transmembrane family of integrins, which are crucial receptors for cell–cell adhesion and also cell adhesion to extracellular matrix proteins, and with their ligands play an irreplaceable role in successful fertilization and embryo development ,3,4. AllThe goal of our study was to identify the precise localization of the αV integrin subunit in intact sperm heads and to investigate its potential changes in membrane reorganization during the acrosome reaction initiated in capacitated sperm. The study targeted αV integrin membrane compartment localization in three species represented by two experimental models with distinct sperm head morphology such as mouse and pig and to compare them with human. A detailed understanding of αV integrin behavior prior to fertilization prepares the ground for future research involving sperm pathologies for the possibility of novel fertility-related protein markers.αV integrin gene expression during spermatogenesis; immunofluorescent protein depiction; super-resolution microscopy, and Western blot detection in sperm and reproductive tissue extracts  strongly covering the apical hook, and lightly covering the post-acrosomal region and neck a–c. In tp < 0.05; *). Using Western blot immunodetection, αV integrin was also determined as a 130 kDa protein in mouse testicular (T) and epididymal (E) tissues. Apart from 130 kDa protein, we observed a signal in a band with a molecular mass of 60 kDa, which is non-specific according to the datasheet for the antibody  and acrosome-reacted (AR) sperm, and 130 kDa protein was detected by immunodetection on the PVDF membrane g. We comantibody g.p > 0.5). The presence of αV integrin (130 kDa) was proven by Western blot analysis in protein extracts from testicular (T) and epididymal (E) tissues  amount of αV integrin (130 kDa) compared to the samples of ejaculated (Ej) sperm  sperm h. Similaj) sperm g.Integrins have been confirmed to be involved in mammalian reproductive processes  includinαV integrin gene expression in the male germ cell subpopulation during spermatogenesis with extended αV integrin protein comparison among three species with distinct sperm head morphology and fertilization behavior. Unlike studies referring to the localization of αV integrin on the IAM [Within this study, we address all the above aspects in detail and delivered comprehensive data regarding  the IAM ,14, we d the IAM .The acrosome reaction is a complex event, which involves massive membrane restructure and protein relocation . MembranThe presence of αV integrin on the sperm surface can have several important roles during fertilization. One of them is the fusion of oviductosomes with the sperm, wherein the interaction is mediated in sperm by integrin heterodimers formed with a αV integrin subunit . ConsideUnless otherwise noted, all chemicals were purchased from Sigma-Aldrich .Inbred C57BL/6 mice were housed in a breeding colony of the Laboratory of Reproduction, IMG animal facilities, Institute of Molecular Genetics of Czech Academy of Science, and food and water were supplied ad libitum. The male mice used for all experiments were healthy, 10–12 weeks old, with no sign of stress or discomfort. All animal procedures and experimental protocols were approved by the Animal Welfare Committee of the Czech Academy of Sciences, Animal Ethics protocol code 66866/2015-MZE-17214, 18 December 2015.cauda epididymis was dissected into smaller pieces and placed into the tube and homogenized. The mouse testis was cut in half, and one half was placed into the tube for following homogenization. Tissues of the testis and cauda epididymis of slaughtered pigs were cut into small pieces and kept at −70 °C for the protein extraction. Then, 10 mg of mouse or porcine tissue of testis or epididymis was placed in the Precellys tubes with beads  and homogenized in 500 µL lysis buffer  by a Precellys tissue homogenizer . Consequently, the samples were centrifuged at 10,000× g, for 10 min, at 4 °C, and the supernatant was transferred into a new tube and was used for SDS-PAGE. Lysate from adult human testis (ab30257-10) and epididymis (ab29975-1) were from Abcam .The whole mouse g, 15 min, 4 °C) and resuspended in TRI Reagent®. The total RNA was isolated according to manufacturer’s instructions and stored at −70 °C.A centrifuge J26XP with an elutriation rotor JE-5.0  were used for obtaining the elutriation fraction. The elutriation protocol was carried out in PBS at 4 °C; the protocol is described in . The celtestis samples using TRI Reagent®. RNA extracts (2 μg) were treated with DNase I  in the presence of DNase I buffer 10 ×  with MgCl2 for 30 min at 37 °C, and EDTA (Fermentas) was added for 10 min at 65 °C. The reverse transcription reaction contained 5 × reaction Buffer (Fermentas), Riboblock Inhibitor (20 U/µL), Universal RNA Spike II , 10 mM dNTP Mix (Thermo Scientific), oligo(dt)18 (Thermo Scientific) mixed 1:1 with Random primers (Thermo Scientific) and M-MuLV RevertAid transcriptase , and run to generate cDNA. All cDNA samples were synthesized in duplicates. The RT- negative control was prepared in the same conditions but with RNase/DNase-free water. For qRT-PCR, 10 ng/µL cDNA was used. Two times Maxima SYBR Green qPCR Master Mix (Thermo Scientific), reverse and forward primer , and nuclease-free water were used, and all reactions were performed in duplets in a PCR cycler . The Ribosomal protein S2 (Rps2) gene was used as the reference gene. Specific gene markers for germinal cells and somatic cells were used to determine elutriation fractions. The RT- negative control for cDNA synthesis was also analyzed.Total RNA was isolated from testicular fractions prepared by elutriation and caudae epididymides were dissected, and sperm from their distal regions were released into a two 200 μL droplets of M2-fertilizing medium (M7167) under paraffin oil  in a Petri dish and pre-tempered at 37 °C in the presence of 5% CO2. Released sperm were assessed for motility and viability under a light inverted microscope with a thermostatically controlled stage at 37 °C with the Computer-Assisted Sperm Analysis (CASA) module and software . In contrary to pig and human, it is not possible to use the ejaculated sperm in the case of mouse. However, in our study, the αV transmembrane protein was investigated, and we do not presume any dramatic changes in this protein behavior during ejaculation.Both g for 10 min at room temperature and were washed twice in PBS, centrifugation 300× g for 10 min at room temperature.Porcine spermatozoa were obtained from Insemination Station Skrsin . Porcine ejaculated spermatozoa were centrifuged from extender medium at 300× ® System  and centrifuged at 300× g for 20 min at 37 °C. Biological materials and experimental protocols were approved by the Ethics Committee of the General University Hospital , protocol code 617/17 S-IV.Human ejaculates were obtained from men after 3–4 days of sexual abstinence, at the Centres for Assisted Reproduction  with the informed consent of the healthy donors and in accordance with the approval of the Ethics Committee, protocol code BIOCEV 012019, 20 January 2019. After liquefaction, the human sperm were separated from seminal plasma by centrifugation gradient (55%/80%), SupraSperm6/mL) were capacitated in 100 µL M2 medium (M7167) for 90 min at 37 °C in 5% CO2. Capacitated status of mouse sperm was determined by staining of anti-phosphotyrosine antibody  [2.Epididymal mouse sperm (5 × 10epublic) . The acr3; 3.2 mM KCl; 0.35 mM NaH2PO4.H2O; 0.5 mM MgCl2.6H2O; 8 mM lactate Ca.5H2O; 10 mM sodium lactate; 5 mM glucose; 2 mM caffeine; 0.17 mM kanamycin sulphate, and 1 mg/mL polyvinyl alcohol). Before using the TALP-stock medium, it was supplemented with 3 mg/mL bovine serum albumin (BSA) and 0.12 mg/mL sodium pyruvate, and the pH was adjusted to 7.4. The porcine sperm (5 × 106/mL) were incubated in capacitated medium for 2 h at 37 °C in 5% CO2. The capacitated status of porcine sperm was determined by staining of anti-phosphotyrosine antibody  according to [2.Capacitated medium for porcine sperm was prepared from TALP-stock medium  were capacitated in sperm preparation medium (Origio) for 2 h at 37 °C in 5% CO2. The capacitated status of human sperm was determined by staining of anti-phosphotyrosine antibody  according to [2 to induce the acrosome reaction.Human ejaculated sperm . Sperm smears were fixed with 2% paraformaldehyde (PFA) for 10 min, which was followed by washing in PBS. After fixation, the sperm were permeabilized by incubation in Intracellular Staining Perm Wash Buffer  3 times for 5 min. Sperm were blocked with 5% BSA in PBS for 1 h and incubated with primary antibody  overnight at 4 °C, which was followed by secondary Goat-anti rabbit antibody  1:300 in PBS for 1 h at room temperature. After the application of the primary and secondary antibodies, sperm were incubated with PNA lectin, Alexa Fluor 568 , diluted 1:500 for 30 min, and consequently incubated with DAPI  for 5 min and washed in PBS. At the end, sperm were washed in distilled water and air-dried. Dry samples were covered with 90% glycerol with 5% anti-fade N-propyl gallate. Multi-color SIM super-resolution images were obtained by DeltaVision OMX™ V4 . An open-source software Fiji  was used2HPO4; 0.49 mM MgCl2; 4.56 mM KCl; 119.8 mM NaCl; 1.3 mM NaH2PO4; 2.37 mM fructose; 14.9 mM NaHCO3 (pH 7.0–7.6). Diluted semen of mouse and human were layered on solution of 1.3 M sucrose with 0.9% NaCl and centrifuged for 30 min at 2000× g at 4 °C. Pellet of sperm was resuspended in 0.15 M NaCl with 5 mM HEPES (pH 7.0), layered on solution of 1.3 M sucrose with 0.9% NaCl, and centrifuged for 20 min at 34,000× g at 4 °C. The sperm pellets were resuspended in 0.15 M NaCl with 5 mM HEPES (pH 7.0) supplemented with protease inhibitors and sonicated  1 × 5 s and 3 × 2 s for mouse, 10 × 10 s intervals for porcine, and 20 × 10 s intervals for human. Membranes from homogenate were separated by a discontinuous sucrose gradient consisting of 1.75 M sucrose and 1.3 M sucrose (1:1) and centrifugation for 3 h at 95,000× g at 4 °C. The plasma membrane (PM) fraction was at the interface between the sample and 1.3 M sucrose solution; the outer acrosomal membrane (OAM) fraction was the interface between 1.3 M/1.75 M sucrose. The pellet contained an inner acrosomal membrane (IAM), and the remaining equatorial segments were closely associated with the sperm heads. The fractions of PM and OAM were diluted with PBS and pelleted by centrifugation for 30 min at 120,000× g at 4 °C. The fraction of IAM was washed by PBS and centrifuged at 3500× g for 10 min at 4 °C. Subsequently, the separate membrane fractions were solubilized with 1% (v/v) Triton X-100 for 1 h at 4 °C. Proteins were precipitated by acetone, and then, samples were incubated in reducing sample buffer for SDS-PAGE at 100 °C for 5 min. A large number of mice would be needed in order to collect the required amounts of epididymal sperm for the membrane fractionation; for ethical reasons, this experiment was not performed.Isolation of sperm plasma membrane and acrosomal membrane was made according to . This me6 sperm were fixated in a mixture of frozen acetone–methanol (1:1). After washing, the sperm pellet was diluted in 80 µL of PBS. The sperm suspension was incubated with PNA lectin, Alexa Fluor 568 ; then, it was diluted 1:500 for 30 min and subsequently with DAPI  for 5 min. Sperm were sorted by BD FACSDiva™  into two fractions: acrosome-intact stained by PNA lectin in the acrosome and acrosome-reacted with PNA stained only in the equatorial segment. Sperm were washed with PBS and centrifuged at 500× g for 5 min. Sperm pellets were dissolved in nonreducing sample buffer for 5 min at 95 °C.After the acrosome reaction, sperm were washed in PBS, and 5 × 10®-P (Merck Millipore). The molecular weight of the proteins was assigned by Precision Plus Protein Dual Color Standards . The membranes were blocked in 5% Blotting-Grade Blocker (Bio-Rad) and incubated with rabbit monoclonal recombinant anti-integrin αV antibody  diluted 1:500 in PBS at 4 °C overnight. After incubation, the membranes were washed in PBS and incubated with secondary antibody, goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad), diluted 1:3000 in PBS for 1 h at RT, and consequently washed in PBS. The membranes were developed with SuperSignal™ Chemiluminescent Substrate (Thermo Scientific). The membrane incubated with rabbit IgG (Invitrogen) with a concentration of 4 μg/mL was used as a negative control.A pellet of mouse , porcine, and human (ejaculated and acrosome-reacted) sperm were dissolved in nonreducing sample solutions with protease inhibitors for 30 min at 4 °C and subsequently boiled for 5 min at 95 °C. The protein extracts from mouse, porcine, and human sperm were separated by 10% SDS-PAGE, and sperm membrane fractions were separated by 12% SDS-PAGE. The proteins from sperm and sperm membrane fractions were transferred onto the PVDF membrane Immobilont-test in GraphPad Prism 5 software  was used for statistical evaluation of densitometric analysis.For all three independent replicates, immunodetection of transferred proteins was performed. Each data point is presented as a mean ± SD. An independent two-sample αV integrin in the individual germ cell subpopulation during spermatogenesis was followed by its protein expression and membrane localization on mouse, porcine, and human reproductive tissue, sperm. Depiction of the exact localization of αV integrin on the sperm head and its novel compartmentalization after the acrosome reaction may presume that αV integrin is a molecule involved in sperm adhesion, interaction, communication with the oviduct, and gamete membrane fusion. The findings relevant to human provide grounds for further research into fertility assessment using αV integrin as a marker for reflecting sperm quality.Delivering positive gene expression of


The shrinkage, chemical composition, and specific surface area (SSA) of samples after heating at different temperatures were analyzed. Our results show that the silicon hydroxyl groups of samples derived from AlCl3·6H2O gradually decreased and nearly disappeared after heating at 800 °C, which indicates the complete dehydration of the silicon hydroxyl. Thus, the samples exhibited a large linear shrinkage and decreased SSA after high-temperature heat treatment. By contrast, samples doped with α-Al2O3 powders retained abundant silicon hydroxyl groups, and the 6.1 wt.% α-Al2O3-doped sample exhibited the lowest linear shrinkage of 11% and the highest SSA of 1056 m2/g after heat treatment at 800 °C. The alumina-doped silica aerogels prepared using a simple and low-price synthesized method pave the way for the low-cost and large-scale production of high-temperature thermal insulation.In this study, we used two methods to prepare alumina-doped silica aerogels with the aim of increasing the thermal stability of silica aerogels. The first method was physical doping of α-Al Silica aerogels are considered important and useful materials due to their nanoporous structure, ultra-low density, low thermal conductivity, high porosity and high specific surface area. Following about 90 years of development, the production of silica aerogels has been industrialized, and they have been widely applied, especially in the thermal insulation field ,2,3,4. O2O3 [2 [2 [2O3 phase and a high SSA of 136 m2/g after heat treatment at 1300 °C. However, the related research remains in the laboratory stage and depends on expensive raw materials and complex processes, making it difficult to carry out large-scale industrial production. Fabricating silica-based aerogels with enhanced heat resistance using an inexpensive process is critical to the development of industrial high-temperature applications. Preparing a silica–alumina composite aerogel represents a potentially effective method for enhancing the heat resistance of aerogels, but the specific synthesis methods  need to be studied. Currently, the precursors of alumina for aerogels mainly include aluminum alkoxides, such as aluminum sec-butoxide (ASB) or isopropoxide (AIP), and alumina inorganic salts such as aluminum chloride (AlCl3·6H2O) and aluminum nitrate (Al(NO3)3·9H2O). Although aluminum alkoxides are the main precursors used to fabricate alumina or alumina composite aerogels, they have some defects that render them unsuitable for industrial production. Aluminum alkoxides have ultra-high reactivity and complex chemical pathways; thus, the reaction process is difficult to control. Moreover, aluminum alkoxides are relatively expensive and are not environmentally friendly. By contrast, alumina inorganic salts are easily controlled during gel preparation and are more economical. Many strategies, including ion-exchange resin, electrosorption ion removal and inorganic ion exchanger [2O3-SiO2 composite aerogels via TEOS and AlCl3·6H2O as precursors, which showed a γ-Al2O3 phase and high SSA of 630.6 m2/g after heat treatment at 600 °C. However, the SSA of Al2O3-SiO2 composite aerogels decreased to 277.7 m2/g due to volume shrinkage after heat treatment at 1000 °C. Chen et al. [2O3-SiO2 aerogels using TEOS and AlCl3·6H2O as precursors and found that the samples with a molar ratio of Al/Si = 3:1 possessed better thermal insulation performance and thermal stability, but the alumina proportion was ultra-high, which was not suitable for industrial-scale production. Lei et al. [2O3 powders. They found that the macropore volume fraction was dramatically reduced from 63.05% to 23.12%, and that the SSA decreased from 837.4 m2/g to 358.5 m2/g with the addition of Al2O3 powders at room temperature; however, they did not mention its high-temperature properties. As using alumina inorganic salts is preferable for industrial production, we selected AlCl3·6H2O and α-Al2O3 powders as the raw materials in low proportions to improve the high-temperature performance of silica aerogels and examined the feasibility of this method for large-scale production.Metal oxides, such as Al2O3 ,16,17,182O3 [2 ,20,21,22O3 [2 [2 ,24,25,26O3 [2 [2  preparedxchanger ,29,30,31xchanger  fabricatn et al.  preparedi et al.  improved3·6H2O and α-Al2O3 powders as the raw materials. We applied two composite methods: Physical doping of α-Al2O3 nano powders and creating a chemical compound through the co-precursor of tetraethyl orthosilicate (TEOS) and AlCl3∙6H2O in low proportions. These methods are low-cost and environmentally friendly as they lack alumina alkoxides and epoxide addition. Furthermore, these methods may promote the industrialization of silica-based aerogels in the application of high-temperature (600–1000 °C) thermal insulation.In this paper, we present an acid-base, two-step sol-gel method for the synthesis of monolithic alumina-doped silica aerogels using AlCl3) and thermal conductivity (0.050 W/m∙K) after calcination at 1000 °C.After heat treatment at 1000 °C for 1 h, most samples retained a monolithic and crack-free macro-morphology as depicted in 2O3. By comparing the two figures, we found that AL samples exhibit lower linear shrinkage before calcination at 800 °C, while AO samples show lower linear shrinkage after calcination at 800 °C.In order to analyze the thermal stability of samples, we focused on the linear shrinkage of all the samples after heat treatment from 300 to 1000 °C with an incremental step of 100 °C . As the −1 indicate the presence of water adsorption [−1 corresponds to an antisymmetric stretching vibration peak of Si–O–Si, and the bands at 798 and 466 cm−1 correspond to a symmetric stretching vibration peak of Si–O–Si. These bands do not show significant changes with the increment of temperature. The bands at 2972, 2898 and 1402 cm−1 at under 400 °C indicate the Si–CH3 vibration of hydrocarbon groups and disappear after heat treatment at over 500 °C. The band at 955 cm−1, which is related to a bending vibration peak of Si–OH, decreases gradually with the increment of temperature and completely disappears after heat treatment at 800 °C. This result indicates a dehydration reaction of Si–OH that results in significant linear shrinkage. For the FTIR spectra of the AO and AL samples after heat treatment at 800 °C, the main peaks were related to water adsorption and the Si–O–Si bond, which are similar to the results for the AL4 sample. However, the band at 955 cm−1 relating to Si-OH reveals different conditions. The peak related to the Si–OH bond of AL samples almost disappears, except for in sample AL2, while the Si–OH bond of all the AO samples still existed after heat treatment at 800 °C, which resulted in a reduced linear shrinkage compared to all the AL samples. The Al–O–Al and AL–OH bonds at 880 and 1074 cm−1 [sorption . The wid074 cm−1 ,37 were 2O3 phase (PDF 10–0173). This was expected because the AO samples were doped with α-Al2O3 powders. However, no AL samples exhibited a diffraction peak, indicating that the abundant silica content inhibited the phase transition of alumina.3·6H2O produces abundant bonds of Al–OH. For AO samples, the situation differed: The addition of α-Al2O3 powders reduced the dehydration reaction of Si–OH, increasing the SSA and decreasing the average pore diameter of AO samples. The AO7 sample, in particular, showed the highest SSA of 1056 m2/g, which is consistent with the lowest linear shrinkage of 11%.Since the AL4 sample showed good thermal stability after being heat treated at 800 °C, we studied the properties of other samples after heat treatment at 800 °C. 3. TEOS, ethanol, deionized water and AlCl3·6H2O reacted according to Equations (1) and (2) and formed abundant Si–OH and Al–OH bonds. During the condensation stage, we adjusted the pH value of the solution to 6–7 using NH4OH. Then, Si–OH and Al–OH bonds reacted according to Equations (3)–(5) and formed abundant Si–O–Si, Al–O–Al and Si–O–Al bonds. There were also residual –OH bonds, especially on the surface of aerogels, which react more at high temperatures. For AO samples, TEOS has the same reactions while α-Al2O3 powders are chemically stable. As the nanoparticles of α-Al2O3 powders have high surface activity, silica primary particles will aggregate on the surface of α-Al2O3 particles through van der Waals forces, the electrostatic force and hydrogen bonding [The sol-gel process is the key step when constructing a robust three-dimensional network of aerogels and can be divided into the hydrolysis and condensation stage. For AL samples during the hydrolysis stage, we adjusted the pH value to 1–2 by using HNO bonding .The specific sol-gel mechanism can be described by Equations (1)–(5) .(a) Hydrolysis.(b) Condensation.2O3 particles. Thus, Si–OH bonds do not react completely at 800 °C and AO samples show less shrinkage and higher SSA.After heat treatment at a high temperature, AL samples have greater shrinkage than AO samples due to the dehydration condensation of –OH, which can be identified by the decrease in Si–OH bonds in the FTIR spectra and the decreased SSA. By contrast, AO samples exhibited better thermal stability due to their stable chemical properties and the crystal phase of α-Al3·6H2O and α-Al2O3 powders as the precursors. Alumina-doped silica aerogels were facilely prepared by two methods: Physical mixing and creating a chemical compound. Although both methods enhanced the thermal stability of silica aerogels before 800 °C, the hydrolysis of AlCl3·6H2O produces Al–OH and promotes the dehydration reaction of Si–OH. This gave the AL samples higher shrinkage and lower SSA after heat treatment at 800 °C. On the contrary, the addition of α-Al2O3 powders reduced the dehydration reaction of the silicon hydroxyl groups, and the 6.1 wt.% α-Al2O3 powders-doped sample exhibited the lowest linear shrinkage (11%) and highest SSA (1056 m2/g) after heat treatment at 800 °C for 1 h. The heat resistance is expected to be further improved through the composition with fiber in order to satisfy industrial requirements. This simple and rapid method can effectively enhance the thermal stability of silica aerogels and facilitate the large-scale production of low-cost thermal insulation applications of silica-based aerogels from 600 to 1000 °C.In this work, we applied an acid-base two-step sol-gel method for the preparation of high-temperature heat-resisting alumina-doped silica aerogels using TEOS, AlCl3·6H2O), ethanol, nitric acid , ammonia  and α-Al2O3 powders (with an average diameter of ≈30 nm) were purchased from Sinopharm Chemical Reagent Corporation . Deionized water was applied in all experiments. All reagents were analytical grade and were used as received without further purification.TEOS, aluminum chloride hexahydrate  = 1, 2, 4, 7, 10% were denoted as AL1, AL2, AL4, AL7 and AL10 for AlCl3·6H2O and AO1, AO2, AO4, AO7 and AO10 for α-Al2O3 powders, respectively.TEOS (30 mL), ethanol (55 mL), and deionized water (5 mL) were added into a glass beaker to obtain solution A. Different proportions of AlClThe wet gels aged for one day and the solvent was displaced with fresh ethanol for 3 days. The ethanol supercritical drying was carried out in an autoclave heated to 260 °C and 10 MPa with a heating rate of 1 °C/min and was held for 1 h at 260 °C. The ethanol in the autoclave was then emitted gradually with a decompression rate of about 2 MPa/h. The obtained alumina-doped silica aerogels were heat-treated at 300, 400, 450, 500, 550, 600, 700, 800, 900 and 1000 °C and the corresponding samples were denoted by adding the heat treatment as a suffix .α radiation . The specific surface area (SSA) and pore size distributions were obtained by a N2 adsorption analyzer (ASAP2460). Thermal conductivity was measured by thermal constant analyzer .The linear shrinkage was calculated depending on the change in the cylinder diameters of the aerogels with an increment in temperature. The samples compared were the as-prepared aerogels derived from super critical drying process without any treatment. Bulk density was determined by the mass and volume of regular cylinders. The surface morphology was observed by scanning electron microscopy . The chemical groups that remained in the samples were investigated by a Fourier transform infrared spectrometer , and all samples were dispersed in dry KBr and pressed into a semitransparent slice for FTIR characterization. The crystal phase of the aerogels was analyzed by powder X-ray diffraction (XRD) with a Rigata/max-C diffractometer using Cu-K


Millettia ferruginea. Compost sample (n = 30) was taken from fresh compost materials and MSW and different organic additive treatments . Compost treatments phytotoxicity test was conducted using lettuce seed (Lactuca Sativa L. var. crispa). Analysis of variance (ANOVA) was performed using SPSS (version 22) on major compost quality characteristics.The present study evaluated the compost quality from municipal solid waste (MSW) and organic additives of coffee by-products and leaf of The compost Physico-chemical characteristics like temperature (26.4 °C), moisture content (45.5%), electrical conductivity (4.6 mS/cm), pH (7.9), total nitrogen (1.2%) and phosphorous content (2918 ppm) in T4 and T5 were analogous but both are significantly different from T3, T2 and T1 compost treatments. Phytotoxicity test using 100% compost treatment media showed that T4 (101%) and T5 (102%) are phytonutrient for lettuce plant. While, T3 and T2; and T1 compost treatments are non-phytotoxic and moderately phytotoxic respectively to lettuce plant.M. ferruginea (T4) and MSW + coffee pulp + M. ferruginea (T5) are important for improving the physico-chemical characteristics of compost and are phytonutrient for lettuce plant. Thus, for effectively management of the 75% of organic fraction of waste generated from households in the study area, recycling methods like composting with organic additives must be used at large.Therefore, compost from MSW + The online version contains supplementary material available at 10.1186/s13065-021-00770-1. Municipal solid wastes are one of the global environmental challenges and major source of environmental pollution . The incIn Ethiopian cities, municipal solid waste is managed using simple and cheapest option like disposing in landfills by means of open dumping . This haMillettia ferruginea (Hochst.) Baker and/or cow dung improve the quality of compost [Coffee by-products are potential threats for environmental quality and human health in coffee growing regions of Southwest Ethiopia, Bench Sheko Zone in particular. For example, every 100 kg of fresh coffee berry gives about 40 kg of wet coffee pulp waste . Poor di compost . Compost compost , 13.Further, organic waste additives are important to facilitate the microbial enzymatic activity, shorten composting period, enhance the quality of matured compost and reduce pollution . SouthweCurrently, compost from municipal solid waste and other organic wastes have been practiced in various parts of globe and showed significant result in waste recovery and soil fertility amelioration , 15, 16.This study was conducted in the tree nursery site at Mizan-Tepi University, Southwest Ethiopia Fig. . The uniFanika solid waste landfill site is located at the lower elevation of Gacheb catchment Fig. . Gacheb M. ferruginea (Hochst) Baker. Municipal solid waste was collected from Mizan–Aman town residential, hotel and market waste landfill sites. Coffee pulp and effluent was collected from nearby wet coffee processing station. M. ferruginea leaves and topsoil were collected from Fanika participatory forest site around Aman town. The collected municipal solid wastes were separated manually using hand into organic, plastics, metals, and inert materials. The MSW composition consists of 80% organic, 10% plastic and 10 others. All organic MSW and leaves of M. ferruginea composting materials were chopped manually into small pieces to aid the microbial activity [The main organic materials for composting are municipal solid waste, coffee by-products and leaves of activity .The composting experiment was done in Mizan-Tepi University at the tree nursery site in July 2019, under shade condition for 70 days. Compost samples were collected from four different points  and those sub-samples were mixed properly for Physico-chemical analysis. All the compost treatments temperature was measured daily using portable compost temperature probe (Reotemp FG20P) at different points . All compost treatments samples were sealed in plastic bag and transported to National Soil Testing Center, Addis Ababa for analysis. All the study samples were analyzed using the standard analytical procedures  proportion in the composting material is important to produce quality compost [3 size , based on pile recommendation for wet region [The compost treatments proportion with additives on dry weight basis was as follows:mentclasspt{minima compost . The comentclass1pt{minimat region . CompostThe compost moisture content was determined as weight loss upon drying in an oven dry (Henan Lanphan DZF6050) at 105 °C to a constant weight . Bulk deTotal nitrogen (TN) was analyzed by Kjeldahl digestion method . Total pLactuca sativa L.) var. crispa in Mizan Tepi University Greenhouse. The experiment was carried out based on complete randomized design (CRD) with three replicates. Soil and compost composite (3:1) and compost (100%) media proportion was used for phytotoxicity test in 5-L plastic pot. Lettuce was selected because of its sensitivity to the presence of phenolic compounds in the growth media and seed germination was inhibited by immature compost [Lactuca sativa L.) var. crispa then at the end of the 2 weeks; lettuce seeds germination and root length were recorded. The variety was selected because of its adaptive to southwest Ethiopia [The matured compost phytotoxicity was determined using seeds of lettuce . The mean difference in physico-chemical characteristics between compost treatments were compared at 95% confidence interval level (p < 0.05). Simple correlation analysis was used to show the correlation of some physico-chemical characteristics of the compost.M. ferruginea (54.9 ± 4.0%) and MSW (49 ± 3.4%) (Table 3) is higher than M. ferruginea (0.17 ± 0.005 g/cm3) and coffee pulp (0.13 ± 0.003 g/cm3). The EC of the MSW (4.1 ± 0.5mS/cm) is higher than coffee pulp (4.0 ± 0.4 mS/cm) and leaves of M. ferruginea (3.9 ± 0.4 mS/cm) (Table The moisture content of coffee pulp (59 ± 4.2%) is higher than leaves of %) Table . Raw comM. ferruginea (6.2 ± 1.0) (Table M. ferruginea (81.4 ± 4.5%). The total nitrogen content of the leaves of M. ferruginea (3.9 ± 0.3%) is higher than coffee pulp (2.7 ± 0.2%) and MSW (1.7 ± 0.2%). The phosphorus content of M. ferruginea (1860 ± 13 ppm) is higher than coffee pulp (1360 ± 15 ppm) and MSW (992 ± 10 ppm). The C:N ratio of MSW (26.2 ± 1.6) higher than coffee pulp (19.0 ± 2.3) and M. ferruginea (12.1 ± 1.3) (Table The pH of coffee pulp 8.9 ± 1.4) is higher than MSW (6.9 ± 0.8) and leaves of .4 is higp < 0.01) from all other treatments (MSW with different organic additives). The presence of high temperature on the compost in T1 may be due to the presence of microbial degradation of organic substrate in the compost heap under T1, as suggested by high C: N ratio compared to other compost treatments. The lower compost temperature was recorded in T5 (26.4 ± 1.0 °C). Similarly, a research reported that temperature fluctuation in the compost pile overtime related to consumption of organics by microorganisms [As illustrated in Table rganisms . In geneHowever, a temperature more than 50 °C was recorded in MSW compost with various organic additives . This suggests that the addition of various organic additives play a significant role in providing optimal C/N ratio to microorganism, and this encourage active microbial biomass, organic matter degradation and rise in temperature  in moisture content between T5 and T1, T2 and T1, T4 and T1, T3 and T1. There was no significant difference between T5 and other treatments of T2, T3 and T4. The presence of high moisture content on treatment amended with different organic additives  may be associated with the presence of relatively high organic matter and low temperature compared to T1. Likewise, other study suggested that organic matter content of the compost increases water holding capacity and ideal moisture content [The highest moisture content was recorded in T5 (45.5 ± 1.3%), followed by T2 (45.1 ± 1.1%) and T4 (45.0 ± 0.9%) (Table  content . Similar content . A resea content . The moi content  and Ethi content . However content  and less content .3), and lowest was recorded at T5 (0.36 ± 0.04 gm/cm3). The compost bulk density in T1 is significantly different (p < 0.05) from T2, T3, T4 and T5. The presence of high bulk density in T1 may be related with the presence of low organic matter in T1. The presence of organic additives like coffee pulp, coffee effluent and leaves of M. ferruginea increased compost organic matter, in turn decreased the bulk density of the compost treatments with organic additives. More importantly, the strong negative correlation (R2 = 0.987) between the bulk density and the organic matter content of the treatment shows the role of additive on the bulk density  [3 [As indicated in Table  , and (0.g. 3) [3 ) of matu+, Ca2+, Na+) and inorganic salts (phosphate and ammonium ions) during microbial decomposition of added organic matter. In general, the reported EC is in-line with the EC content (1.5–8.8 mS/cm) [As shown in Table 8 mS/cm) , 4.2–5.28 mS/cm)  and 2.2–8 mS/cm)  of matur8 mS/cm) .p < 0.05) in between T5 and T1, T5 and T2; and T5 and T3. However, no significant difference was recorded in pH of the compost between T5 and T4. The presence of high pH in T5 and T4 may be related with the release of base cations like, K+, Ca+, Mg2+ and Na+ during microbial biodegradation of various organic additives like coffee pulp, coffee effluent and M. ferruginea. Similarly, a study report showed that the increase in pH of the compost is attributed to the releases of base cation during decomposition of various organic additives [As indicated in Table dditives . The pH dditives , and beldditives . Furtherdditives –42 and pdditives .Table 3Pp < 0.05) between T5 and T4, T5 and T3, T5 and T2 and T5 and T1. Similarly, organic matter content of T4, T3 and T2 are significantly different from T1. The presence of high organic matter in T5 may be related with the presence of organic additives like coffee pulp and M. ferruginea. Also, the presence of relatively high organic matter in T2, T3 and T4 associated with the presence of additives like coffee effluent, coffee pulp and M. ferruginea respectively. The organic matter content of these treatments was in line with the organic matter content of German compost quality standards (> 15%), Austrian (> 20%) and South Africa (20–50%) and organic matter (17–22%) of matured compost [The highest organic matter content of the compost was recorded in T5 (25.6 ± 0.8%) Table . There wndards > %, Austri compost  and Ethi compost  and orga compost , 38.p < 0.05) between T4 and T3, T4 and T2; and T4 and T1. The presence of high total nitrogen in T5 and T4 may be related with the presence of M. ferruginea in those treatments. This study result is in-line with the research reported an increases in nitrogen content in soil supplemented with leaves of M. ferruginea [The highest total nitrogen content was recorded in T4 (1.2 ± 0.05%) and T5 (1.2 ± 0.05%) Table . There wrruginea . The nitrruginea , 45.p < 0.05) in available phosphorus content between T5 and T1, T5 and T2, T5 and T3. The presence of high available phosphorus in T5 and T4 may be related with the releases of available phosphorus during microbial degradation of organic additives like coffee pulp and M. ferruginea. Similarly, a research study reported that leaves litter of M. ferruginea and coffee pulp increases available phosphorous content of the soil [Table the soil , 46. Furthe soil . The finthe soil .p < 0.05) in C:N ratio between T1 and T2, T1 and T3, T1 and T4; and T1 and T5. The presence of high C:N ratio in T1 may be related with low microbial degradation because of imbalance in C:N ratio on composting material. The C:N ratio of all the treatments were in agreement with the C:N ratio (< 25:1) of stable compost [The highest C:N ratio was noted in T1 14.3 ± 0.4), while the lowest was recorded in T4 (11.3 ± 0.3)  showed that the high germination index was recorded on T5 (102%), followed by T4 (101%), T3 (82%), T2 (82%) and T1 (72%)  do not have phytotoxicity effect and are an important phytonutrient for lettuce plant. However, only T5 and T4 are an important phytonutrient for lettuce plant in 100% compost media.M. ferruginea separately and in combination (MSW + coffee pulp + M. ferruginea) is important for compost quality amelioration and production of phytotoxic free compost. Most importantly, T4 (MSW + M. ferruginea) and T5 (MSW + coffee pulp + M. ferruginea) have higher compost quality and are important phytonutrient for lettuce plant. Amending soil media with compost treatments in 3:1 is important for lettuce seed germination, root elongation and overall soil media nutrient amendment. Since, southwest Ethiopia soil is acidic in nature, the presence of high EC on the compost treatments are important for amelioration of soil acidity problem in the region.Municipal solid waste (MSW) composting with other locally available organic additives like coffee pulp, coffee effluent and Thus, for effectively management of the 75% of organic fraction of waste generated from households in the study area, recycling methods like composting with organic additives must be used at large. Hence, municipality must work towards waste recycling and composting as waste reduction measures. Therefore, municipality must create awareness to society regarding separation of organic waste from altogether, and recycling of waste by composting.Additional file 1: Field experimental photos of composting treatmentAdditional file 2: Compost dataset and analysis


Bankia setacea . These strains, designated as Bs08T, Bs12T and Bsc2T, are Gram-stain-negative, microaerobic, gammaproteobacteria that grow on cellulose and a variety of substrates derived from lignocellulose. Phenotypic characterization, phylogeny based on 16S rRNA gene and whole genome sequence data, amino acid identity and percentage of conserved proteins analyses, show that these strains are novel and may be assigned to the genus Teredinibacter. The three strains may be differentiated and distinguished from other previously described Teredinibacter species based on a combination of four characteristics: colony colour , marine salt requirement , the capacity for nitrogen fixation (Bs08T and T. turnerae strains) and the ability to respire nitrate (Bs08T). Based on these findings, we propose the names Teredinibacter haidensis sp. nov. (type strain Bs08T=ATCC TSD-121T=KCTC 62964T), Teredinibacter purpureus sp. nov. (type strain Bs12T=ATCC TSD-122T=KCTC 62965T) and Teredinibacter franksiae sp. nov. (type strain Bsc2T=ATCC TSD-123T=KCTC 62966T).Here, we describe three endosymbiotic bacterial strains isolated from the gills of the shipworm,  Cellulose is considered to be the most abundant biological material on Earth  and numeTeredinidae (shipworms) are among the best-known hosts of cellulolytic bacteria [In marine environments, bivalves of the family bacteria . Shipworbacteria . Unlike bacteria . Insteadbacteria . Shipworbacteria .Teredinibacter turnerae and Teredinibacter waterburyi are microaerophilic cellulolytic bacteria placed within the family Cellvibrionaceae [Teredinidae [Bankia setacea [Thiosocius teredinicola 2141TT has been isolated from the gills of the giant mud-dwelling shipworm Kuphus polythalamius, indicating that non-cellulolytic bacteria can also form symbiotic partnerships with teredinids [Shipworm gill endosymbionts are unique in that they are among the few intracellular endosymbionts that have been grown successfully in pure culture . To dateonaceae . The foredinidae , while t setacea . Consist setacea . In addiredinids T. waterburyi, three similar endosymbiont strains, designated Bs08T, Bs12T and Bsc2T were isolated from feathery shipworm Bankia setacea [B. setacea gill community [Teredinibacter haidensis sp. nov., Teredinibacter purpureus sp. nov. and Teredinibacter franksiae sp. nov., with Bs08T, Bs12T and Bsc2T, as their respective type strains.In addition to  setacea . Togetheommunity . In thisT, Bs12T and Bsc2T were isolated from the gill and cecum of the shipworm B. setacea collected from the Puget Sound, Washington State, USA as described by O’Connor et al. [et al. [4Cl. Plates were incubated at either 18 or 30 °C until individual colonies could be observed. To rapidly identify novel bacteria on the motherplates, 16S rRNA genes were amplified directly from the colonies using Phire Animal Tissue Direct PCR Kit (Finnzymes F-170S) using 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) primers [Strains Bs08r et al.  and Alta [et al.  at pH 7.T was isolated from a motherplate inoculated with gill homogenate and incubated at 30 °C. A purple colony growing on a motherplate incubated at 18 °C was purified and designated as strain Bs12T. Strain Bsc2T was isolated from a motherplate inoculated with cecum homogenate and incubated at 18 °C.Strain Bs08T is identical in sequence to that of symbiotic bacteria localized to the gills of B. setacea by metagenomic sequencing and fluorescent in situ hybridization [T was shown to be abundant in gills of B. setacea but has not been detected in cecum by in situ hybridization techniques [B. setacea has previously been shown to be largely devoid of bacteria [T in cecum homogenates was likely due to contamination by neighbouring gill tissues during dissection.The 16S rRNA gene of Bsc2dization  and to tdization . Becausechniques  and beca4Cl in test tubes (18 mm × 150 mm) and shaken at 100 r.p.m. in an incubator set to 20 °C. Preparation and revival of frozen stocks are detailed in Altamia et al. [For all the subsequent experiments, unless specified otherwise, strains were propagated in 6 ml of liquid SBM medium with either 0.2 % cellulose (w/v) or 0.5 % galactose (w/v) with 0.025 % NHa et al. .4Cl or 5 mM NaNO3 with the following carbon sources and concentrations (% w/v): polysaccharides—cellulose (0.2 %), carboxymethylcellulose (0.5 %), d-galacto-d-mannan from locust bean (0. 1%), β-d-glucan from barley (0.1 %), lichenan (0.1 %), starch (0.1 %), pectin (0.1 %), dextran (0.1 %), curdlan (0.1 %), pachyman (0.1 %), laminarin (0.1 %), pullulan (0.1 %), chitin (0.1%) and chitosan (0.1 %); sugars—cellobiose (0.5 %), xylan (0.5 %), sucrose (0.5 %), xylose (0.5 %) arabinose (0.5 %), galactose (0.5 %), glucose (0.5 %); organic acids—formate (0.1 %), acetate (0.1 %), propionate (0.1 %) and pyruvate (0.1 %); and amines—N-acetylglucosamine (0.1 %), gelatin (0.1 %), yeast extract (0.1 %) and casein hydrolysate (0.1 %). All strains grew on cellulose, galactomannan, β-d-glucan, lichenan, pullulan, gelatin and casein hydrolysate and all sugars tested. Strains Bs08T, Bs12T and Bsc2T and previously described Teredinibacter species do not hydrolyse agar, distinguishing them from the closely related Saccharophagus degradans 2-40T [Agarilytica rhodophyticola strain 017T [The ability of the strains to utilize carbon substrates was tested by transferring colonies to SBM liquid medium supplemented with 0.025 % NHs 2-40T  and Agaain 017T . None ofT was found to be capable of nitrogen fixation. These results are supported by analyses of their respective genomes which indicate that only Bs08T encodes genes of the Nif operon which is diagnostic of nitrogen fixation [Lyrodus pedicellatus fix nitrogen in situ within host cells, suggesting symbiont diazotrophy might help supplement their nitrogen deficient diet of wood [T. turnerae T7902T, Bs08T and the thioautotrophic shipworm symbiont Thiosocius teredinicola 2141TT) and three  are incapable of fixing atmospheric dinitrogen. This indicates that although diazotrophy may be a common feature of shipworm gill endosymbionts, it is not a prerequisite for endosymbiotic existence. Notably, diazotrophy is not common among described species of the family Cellvibrionaceae. Of these, only Cellvibrio diazotrophicus [T. turnerae [nifH homologs have been found in the genomes of Agaribacterium haliotis feces2T and in the as-yet undescribed Pseudomaricurvus sp. HS19.The diazotrophic ability of these strains was tested by re-streaking colonies on SBM medium with 0.2 % cellulose but without an added nitrogen source. Of these three strains, only Bs08fixation . It has  of wood . Interesurnerae  have bee3 were re-streaked on a solid version of this medium prepared with 1.0 % Bacto agar. Control plates containing SBM medium with 0.2 % cellulose and 0.0025 % NH4Cl were also used. The inoculated plates were then placed inside an anaerobic pouch (BS GasPak EZ Anaerobic Pouch). Despite the presence of homologs of dissimilatory nitrate reductase (nar) in the genomes of all three strains, only strain Bs08T grew under anaerobic conditions in the presence of nitrate.The ability of these three strains to respire nitrate was also tested. Briefly, cells grown in liquid SBM medium with 0.2 % cellulose and 5 mM NaNO4Cl) with liquid cultures using sterile needles. For all strains, a faint lens of cells forms a few millimetres below the soft agar surface, indicating that, like T. turnerae and T. waterburyi Bs02T, strains Bs08T, Bs12T and Bsc2T, show increased growth under microaerobic conditions. Also, like T, turnerae, clearing of the medium was observed around the growth lens after prolonged incubation, due to the hydrolysis of cellulose.To determine the oxygen growth requirements of the three strains, stab cultures were prepared by inoculating soft agar tubes (18 mm×150 mm tubes containing 15 ml of 0.2 % Bacto agar (w/v), 1 x SBM medium, 0.2 % cellulose and 0.025 % NH4Cl as previously described [T. turnerae T7902T, strains Bs12T and Bsc2T, require concentrations of Ca+2 and Mg+2 in the medium similar to those found in seawater (~10 mM Ca and 50 mM Mg), while T. waterburyi Bs02T and strain Bs08T do not.Growth optima, ranges and doubling times were determined in an artificial seawater-based SBM liquid medium supplemented with 0.5 % galactose (w/v) and 0.025 % NHescribed . The dat4Cl growing at 20 °C and submitted to midi  for analyses. The three major fatty acids for all three strains are C15 : 0 iso, C15 : 0 anteiso and C17 : 0 anteiso. Strains Bs12T and Bsc2T have very similar FAME profiles wherein C15 : 0 anteiso and C17 : 0 anteiso account for more than 80 % of fatty acids detected. In strain Bs08T, the total for these two fatty acids is less than 50 %. FAME profiles of these three B. setacea endosymbionts are markedly different from recently described B. setacea symbiont T. waterburyi Bs02T which has dominant fatty acids composed of summed feature 5 . This is consistent with its more divergent phylogenetic relationship as indicated in Cellular fatty acid methyl ester (FAME) analysis was performed using the MIDI Sherlock Microbial Identification System. Briefly, week-old colonies were scraped from SBM medium agar plates supplemented with 0.5 % galactose and 0.025 % NHT, Bs12T and Bsc2T, growing on SBM plates supplemented with cellulose initially appear as minute, slightly raised and translucent. Upon prolonged incubation, the colonies widen in the subsurface of the agar and produce a characteristic clearing zone due to the hydrolysis of cellulose. At this stage, colonies of strains Bs08T and Bsc2T appear off-white or beige, while those of strain Bs12T develop a deep-purple colour. Purple pigmentation is also observed in the culture supernatant when strain Bs12T was grown in liquid medium. No similar pigment production was observed in Bs08T and Bsc2T cultures or in previously described Teredinibacter species. Morphology of cells at mid-exponential phase in liquid medium was examined using phase contrast light microscopy (Nikon Eclipse Ni-U and Nikon NIS Elements). The cells of all the three strains appear as motile slightly curved rods that are 2–5 µm long and 0.4–0.6 µm wide, and are Gram-stain negative (Sigma-Aldrich).Colonies of strains Bs08et al. [et al. [T and Bsc2T were sequenced at New England Biolabs using PacBio Model RSII platform, while the genome of strain Bs12T was sequenced by the Joint Genome Institute – Department of Energy (JGI-DOE) using PacBio Model RS II. All the genomes were assembled using HGAP version 2.0. Contigs from previously reported genome sequence data for these strains [Genomic DNA extracts used for whole genome sequencing and 16S rRNA gene sequence amplification were obtained from bacterial cell pellets grown on SBM liquid medium with 0.2 % cellulose or 0.5 % galactose using the method described in O’Connor et al.  and Alta [et al. . The gen strains  were aliT is composed of a single circular chromosome of 4 915 572 bp with a G+C content of 47.2 mol%. The estimated sequencing coverage is 114×. The genome of strain Bsc2T was assembled to form nine scaffolds totaling to 5 406 307 bp with a G+C content of 47.31 mol% with estimated coverage of 62×. The assembled genome of Bs12T is composed of 2 circular elements, a chromosome of 4 643 613 bp with a DNA G+C content of 45.89 mol% and a large plasmid of 249 467 bp with a DNA G+C content of 42.54 mol%. Estimated genome coverage is 210×. The cumulative GC-skew plots calculated for the two elements are typical of closed-circular DNA structures [https://db-mml.sjtu.edu.cn/ICEfinder/ICEfinder.html), differs from the chromosome both in mol% G+C and in tetranucleotide frequency. Additionally, it contains a copy of recA, a gene most commonly found as a single copy in bacterial chromosomes [recA copy is most similar to that found in the chromosome of Marinobacter confluentis, sharing 79.52 % identity at 95 % query coverage with this recA as compared to 75.9 % identity at 95 % query coverage with the recA copy found in the Bs12T chromosome. These data suggest that this plasmid was acquired recently via lateral gene transfer. Detailed analysis of the genomes of strains Bs08T, Bs12T and Bsc2T will be reported elsewhere. A table comparing the genomes of strains Bs08T, Bs12T and Bsc2T and related bacteria are shown in The assembled genome of strain Bs08ructures   . Phylogenetic relationships among taxa were then inferred using RaxML 8.2.12 [protgammablosum62 substitution matrix and 250 bootstrap replicates were performed to generate a consensus tree. The resulting topology provides strong support for a clade containing strains Bs08T, Bs12T and Bsc2T and previously described species of the genus Teredinibacter. The same analysis places Cellvibrionaceae bacterium PMS-1162T.S.0a.05 basal to this clade, although with more modest bootstrap support.  values less than 85 and 60 % have been suggested as thresholds for delimitation of species and genera respectively . Similaret al.  and Qin  [et al.  respecti–70.18 %  and POCP–70.18 % . These vTeredinibacter turnerae.The description is as given by Distel et al. 2020  with theTeredininbacter haidensis .4Cl or nitrate for growth under normoxic conditions. Has the ability to perform anaerobic nitrate respiration when grown in liquid or solid medium supplemented with cellulose as the sole carbon source. When grown on plates, small translucent colonies are initially observed on the agar surface, but over time the majority of growth forms an inverted dome of cells with off-white or beige coloration beneath the agar surface. The pH, temperature and salinity range for growth is approximately pH 6.5–8.5, 15–30 °C and 0.1–0.6 M NaCl, respectively; with optimum growth recorded at pH 8.0, 20 °C and 0.3 M NaCl. Under these conditions, the observed mean doubling time is approximately 5.2 h. The major fatty acids are C15 : 0 anteiso, C15 : 0 iso and C17 : 0 anteiso. The genome is estimated to be 4.92 Mbp. The G+C content is 47.2 mol%. The GenBank/EBML/DDBJ accession numbers for the bioprojects, chromosomal sequences and 16S rRNA genes for Teredinibacter haidensis sp. nov. Bs08T are PRJNA340180, CP060084 and MT416119, respectively. The type strain has been deposited as T. haidensis Bs08T (=ATCC TSD-121T=KCTC 62964T).In addition to the characteristics of the genus, capable of fixing atmospheric dinitrogen under microaerophilic growth conditions. Can use NHTeredininbacter purpureus (pur.pu′re.us. L. masc. adj. purpureus purple).4Cl or nitrate for growth. When grown on plates, small translucent colonies are initially observed on the agar surface, but over time the majority of growth forms an inverted dome of cells with pronounced purple coloration beneath the agar surface. The pH, temperature and salinity range for growth are approximately pH 6.5–8.5, 10–25 °C and 0.2–0.6 M NaCl, respectively; with the optimum growth recorded at pH 8.0, 20 °C and 0.4 M NaCl. Under these conditions, the mean doubling time is approximately 8.8 h. The major fatty acids are C15 : 0 anteiso, C17 : 0 anteiso and C15 : 0 iso. The total genome is estimated to be 4.89 Mbp and is composed of two closed circular elements, a chromosome of 4 643 613 bp with a DNA G+C content of 45.89 mol% and a large plasmid of 249 483 bp with a DNA G+C content of 42.54 mol%. The GenBank/EBML/DDBJ accession numbers for the bioproject, chromosomal sequence, plasmid sequence and 16S rRNA genes for Teredinibacter purpureus sp. nov. Bs12T are PRJNA195847, CP060092, CP060093 and MT416120, respectively. The type strain has been deposited as T. purpureus Bs12T (=ATCC TSD-122T=KCTC 62965T).In addition to the characteristics of the genus, requires a source of combined nitrogen such as NHTeredininbacter franksiae (frank′si.ae. N.L. gen. n. franksiae named in honour of microbiologist Diana Franks for her contributions to the characterization of the genus Teredinibacter).4Cl or nitrate for growth. When grown on plates, small translucent colonies are initially observed on the agar surface, but over time the majority of growth forms an inverted dome of cells with off-white or beige coloration beneath the agar surface. The pH, temperature and salinity range for growth is approximately pH 6.5–8.5, 15–20 °C and 0.2–0.4 M NaCl, respectively; with the optimum growth recorded at pH 8.0, 20 °C and 0.2 M NaCl. Under these conditions, the mean doubling time is approximately 11.5 h. The major fatty acids are C15 : 0 anteiso, C17 : 0 anteiso and C15 : 0 iso. The total genome is estimated to be 5.41 Mbp with a G+C content of 47.3 mol%. The GenBank/EBML/DDBJ accession numbers for the bioprojects, chromosomal sequences, 16S rRNA genes for Teredinibacter franksiae sp. nov. Bsc2T are PRJNA655743, JACJUV000000000, and MT416121, respectively. The type strain has been deposited as T. franksiae Bcs2T (=ATCC TSD-123T=KCTC 62966T).In addition to the characteristics of the genus, requires a source of combined nitrogen such as NHClick here for additional data file.