Isolates A1 Research Articles

Isolasi dan Uji Aktivitas Antagonisme Jamur Endofit Tanaman Pisang (Musa paradisiaca L.) terhadap Fusarium oxysporum

Fungi that grow in plant tissue without harming the host are called endophytic fungi. This fungus has the potential as a biological agent (biocontrol) in controlling plant diseases. One of the fungi that can cause disease in banana plants is Fusarium oxysporum, which can cause Fusarium wilt disease. Endophytic fungi have antagonistic properties in the form of nutritional competition and antibiosis. These antagonistic properties can be used as biological agents. The purpose of this study was to isolate and test the antagonistic activity of the endophytic fungus of banana plant (Musa paradisiaca L.) against the pathogenic fungus Fusarium oxysporum. This research is an experimental study, which begins with the isolation of endophytic fungi and then continues with the antagonism activity test. The results of the isolation obtained 4 isolates of endophytic fungi, namely: 1) isolate D1 (Phytium sp.); 2) isolate B1 (Aspergillus sp.); 3) isolate A1 (Trichoderma sp.); and 4) isolate A2 (Penicillium sp.). Based on the antagonism activity test, isolate A1 showed the best results in inhibiting the fungus Fusarium oxysporum through the mechanism of space and nutrient competition, while isolates that showed antibiotic activity were isolate A2. The results of the inhibition test using the dual culture method showed that, 2 isolates (B1 and A2) could inhibit the growth of the fungus Fusarium oxysporum. The highest percentage of inhibition was owned by isolate A2. Based on its ability to inhibit Fusarium oxyaporum, endophytic fungal isolates have the potential to be further developed as Fusarium oxysporum control agents.

DEVELOPMENT OF BIOINSECTICIDES AGAINST SPODOPTERA FRUGIPERDA USING CHITINOLYTIC BACTERIA

Actinomycetes play an important role in the biological control of insects by producing insecticidal active compounds against various insects. They are predominantly found in soil, in the silt of water bodies, in the air and in plant remains. Chitinase is increasingly finding applications in various fields such as biomedicine, in ultra-structural studies, in the preparation of chitooligosaccharides, in single-cell protein production, biocontrol agents, agriculture, etc. Chitinase is originally an enzyme used by insects to degrade the structural polysaccharide of insects. Chitinase brings about hydrolysis of chitin which is widely distributed in cell wall of fungi, plants and insects. An attempt has been made to use chitinase producing bacteria for the development of bioinsecticides against Spodoptera frugiperda, which feeds on over more than 60 species of plants and it is periodic. The Spodoptera frugiperda armyworm (Lepidoptera) is a serious pest of cabbage and other crop. It is also major pest of maize, rice, sorghum, turf grasses, cotton, and peanuts, etc. In this attempt enrichment and isolation of chitinase producing bacteria from soil was carried out. Chitinolytic activities of the three isolates were tested against larvae of Spodoptera frugiperda. Three isolates were individually applied against Spodoptera frugiperda. The isolates named Ai, A2 and A3 were found to be more effective against Spodoptera frugiperda. The insecticidal activity of isolate A1was tested and control within 5 hours. Isolate A2 was treated with larvae Spodoptera frugiperda killed after 8 hours whereas, isolate A3 controlled after 24 hours. The evaluated insecticides provided a significant reduction in the infestation level, for variable periods.

The Effectivity of Indigenous Rhizobacteria and Manure on the Yield of Red Potato (Solanum tuberosum L.)

Potatoes are one of the world's five food crops in the form of tubers and have many benefits. One type of potato developed in Indonesia is red potatoes because they have a high nutritional content and are more susceptible to plant pest organisms. An experiment was conducted in Nagari Selayo Tanang Bukit Sileh, Lembang Jaya, Solok, West Sumatra, Indonesia from May to August 2017. The effect of three indigenous rizobacteria (A2.1b2, A3.1a5 and B1.2a2) and three types of manure, both individually and in combination, on the growth and yield of red potatoes was studied. A two way factorial experiment with a completely randomized design and three replicates was used. Chicken, quail and cattle manure were applied at a dose of 30 tons per hectare. Data were analyzed by analysis of variance and comparison of mean using the Honestly Significant Difference test at the 5% level. There was no interaction between rizobacterial isolates and manure on the growth and yield of red potatoes. All isolates gave similar results with respect to plant growth, but isolates A2.1b2 give the best yields. Chicken manure increased fresh weight of tubers by 11.73 ton per hectare compared to quail and cattle manure.

Activity of bacterial seed endophytes of landrace durum wheat for control of Fusarium foot rot

Five bacterial endophytic isolates obtained from durum wheat seeds (landrace “Timilia reste nere”) and identified as belonging to Pantoea (isolates A1, F7, F15 and G1) and Paenibacillus (isolate B) genera on the basis of 16S rDNA gene sequences, were assayed in vitro and in vivo for their ability to inhibit Fusarium culmorum growth and the disease (Fusarium foot rot) it causes in durum wheat. All isolates significantly reduced in vitro growth of F. culmorum in comparison with the control. After 120 hours of incubation, isolates B and G1 showed the greatest mycelial growth inhibition, i.e., respectively, 76 and 74%. When durum wheat “Simeto” seeds were treated with bacterial isolates singly or in combinations and then inoculated with F. culmorum, all treatments with endophytes showed increased, but not statistically significant, seed germination. Except for isolate A1, all bacterial isolates stimulated vegetative parameters of durum wheat seedlings. Mixture of isolates F7 + F15 was the most effective in improving shoot height (+94%), root length (+47%) and vigour index (+81%). Mixture of isolates A1 + B reduced Fusarium foot rot incidence (-21%) and severity (-30%), and isolate A1 reduced incidence (-15%) and severity (-16%) of the disease. These results indicate potential of bacterial seed endophytes, identified in this study, for control of Fusarium foot rot and suggest that bacterial seed endophytes may provide a new biocontrol agent for an environmentally sustainable durum wheat disease management programme.

Isolation and molecular characterization of domestic Bradyrhizobium species from soybean roots in the savannah soil of Nigeria

Abstract. The identification of effective indigenous strains of Bradyrhizobia could lead to the development of efficient and affordable inoculants for improving soil fertility. This can also promote nitrogen fixation in smallholder farming systems of Nigeria and as well make the use of nitrogen fertilizers unnecessary. This study was conducted to characterize and evaluate the nodulating properties of indigenous Bradyrhizobium species in soybean plant. A total of 18 strains were isolated using Bradyrhizobium japonicum selective medium (BJSM) from the root nodules of plants harvested from five sites on Ahmadu Bello University farm, Zaria Kaduna State, Nigeria. These isolates were evaluated for nodulating potential in a screen house using soybean (TGx 1448-2E) as a test crop. The total number of nodules, percentage effective nodules, nodule fresh and dry weights, shoot fresh and dry weight and plant nitrogen content were assessed. The nodules produced by the isolates showed high percentage effectiveness with isolate A4 having significantly higher nodule dry weight (78.00 mg) than the rest of the test isolates. The soybean inoculant, BIOFIX used in this study performed poorly having a nodule dry weight of 6.0 mg. There was positive and highly significant correlation between the nodule dry weight, shoot dry weight and plant nitrogen content (r=0.740, 0.641, 0.616, respectively) at p<0.001. Sequence analyses were carried out on the high performing isolates from each site of sampling and the result obtained using the NCBI Database showed similarity of these isolates with reference strains belonging to the genus Bradyrhizobium (A4- Bradyrhizobium japonicum RV9, B2- Bradyrhizobium guangdongense CCBAU 51649, C1- Bradyrhizobium sp. UFLA05-149, D3- Bradyrhizobium sp. B918 and E3- Bradyrhizobium sp.UFLA05-149). A 16srRNA phylogenetic tree constructed with the sequences obtained grouped the isolates without any close reference strain. However, isolate B2 showed close affiliation with Bradyrhizobium guagdongense with 95% sequence identity. Based on the effectiveness of these five strains of Bradyrhizobium, it is suggested that they can be used as potential candidate for inoculants production.

Screening of amilolytic bacteria in the digestive tract of Red Snapper (Lutjanus campechanus) as probiotic candidates

Fish utilize energy from carbohydrate sources in small amounts because of the low secretion of the enzyme amylase for digestion. One alternative is to increase the availability of exogenous digestive enzymes by utilizing bacteria from the digestive tract that have amylolytic activity. This study aims to isolate amylolytic bacteria in the digestive tract of Red Snapper as a probiotic candidate. This study used several methods, namely amylolytic test on starch, test for resistance to acid conditions (pH 3), observation of bacterial growth for 30 hours, antagonistic test against pathogenic bacteria, test for attachment to the stomach, gram staining test, and pathogenicity test on shrimp. Screening of amylolytic bacteria in the digestive tract of red snapper produced five isolates, namely A1, A2, A3, A4, and A5. From the results of the amylolytic test, only three isolates were further tested, namely A1, A2, and A4. The three isolates were able to survive in acidic conditions of pH 3 and pH 7 for eight hours with density values ​​(OD) in isolates A1 (0.630), A2 (0.597), and A4 (0.601). Isolates A1, A2, and A4 respectively produced antagonistic values ​​with inhibition zones of 8.4 mm, 7.5 mm, and 10.3 mm. Next. the ability to attach bacteria to isolates A1 (6.6 x 102 cfu/ml), A2 (11.8 x 102 cfu/ml) and A4 (16.5 x 102 cfu/ml). After that, the pathogenicity test was carried out aimed at proving that the isolate was not pathogenic to the host. Based on the results of the study, there were three bacterial isolates (A1, A2, and A4) in the digestive tract of red snapper that had met the requirements for probiotic candidates in fish/shrimp and the best was A4 isolate.

A comparative study of xylanase producing wild and mutated strains of Streptomyces variabilis VITMUVB02 isolated from Kanyakumari salt pan

The current study aimed to isolate xylanase-producing marine actinobacteria, which is a novel idea; ascribed to the challenges associated with the culturing of marine microorganisms. A total of 5 actinobacterial colonies were isolated from Kanyakumari marine sediments. Among them only one isolate, A1 indicated the elevated xylanase activity manifesting a zone of hydrolysis of around 20 mm during primary screening. On the other hand, secondary screening of A1 i.e., sugar estimation by 3,5-dinitrosalicylic acid method, resulted in A1 exhibiting 400 µg/ml of enzyme activity. The enzyme extracted from the isolate A1 also contained a protein concentration of 600 µg/ml. Physical and chemical mutation studies were carried out to over-express xylanase production. Chemical mutation involving the use of EDTA did not show a significant increase in xylanase production and thus, was not subjected to further analyses. The isolate, A1 also showed a zone of hydrolysis of 28 mm in the physical mutation study and was named A'1.75. This zone was 8 mm more than the wild-type strain after UV-exposure for 75 min. A'1.75 also exhibited an enzyme activity of 600 µg/ml and protein content of 800 µg/ml. The potential isolate was identified as Streptomyces variabilis VITMUVB02 using 16S rRNA molecular sequencing. The molecular weight of the purified xylanase extracted from A1 and A'1.75 was found to be 25 kDa and 20 kDa, respectively. Fourier Transform Infrared spectroscopy (FT-IR) and High-Performance Liquid Chromatography (HPLC) analyses showed the mutagenic effect with the change in the spectral and functional groups.

Isolation and identification of nitrifying bacteria from tilapia (Oreochromis sp.) pond in Sleman Yogyakarta Indonesia

This research aims to isolate and identify autochtonous nitrifying bacteria from tilapia pond in Sleman Yogyakarta Indonesia for future application in aquaculture practices in the region. Bacteria were isolated using a nitrification medium. Bacterial characterization was carried out by non-pathogenic test to tilapia (Oreochromis sp.), and nitrification activity test in a single bacterial fermentation medium for 9 days. Bacterial identification was carried out based on the colony and cell morphologies, biochemical tests, and molecular analysis using the 16S rRNA and gyrB genes. A total of 15 isolates of nitrifying bacteria were obtained. Four non-pathogenic isolates obtained the highest nitrification activity on the sixth day of incubation, with nitrate production of 17.26-21.54 ppm. Two selected bacteria, isolates A2 and A3, have colony morphology that is milky white, smooth surface, circular shape, entire edge, and convex elevation. Both bacteria are short rods, Gram-negative, non-motile, produce catalase, fermenting glucose, sucrose, and lactose, and do not produce oxidase, ornithine decarboxylase, indole, and H2S. Molecular analysis showed that the two isolates had the highest similarity (99.28% and 99.34%) to Klebsiella spp.

Isolation, Optimization and Application of Pigment Producing Bacteria from Soil

Color is the most crucial aspect of any item since it improves its attractiveness and acceptability. Different dyes are available in the market which makes their way to different industries. Synthetic colors are more reliable and less expensive than natural colors, but due to health hazards, few remain prohibited. In dye, food, pharmaceutical, cosmetic industries, widely use innocuous pigments from microbes. The work focuses on isolating the pigment-producing bacteria from soil and investigate several parameters that influence pigment synthesis. Bio colorant derived from A1 and A2 were applied on cotton fabric with pigment extracted by the solvent method. Different temperature, pH, incubation, and static and shaking conditions were investigated in this study. Biochemical and Molecular analysis identified both isolates A1 and A2 as Pseudomonas aeruginosa and SalinococcusI roseus. This work demonstrated pigment production was good under neutral pH and at shaking conditions. Both isolates showed different incubation times for pigment synthesis. Only Pseudomonas aeruginosa exhibited antibacterial action against the test pathogens E.coli and Staphylococcus aureus.

Inhibition of biofilm formation of Geobacillus stearothermophilus in calcium-reduced milk protein formulation is associated with calcium, sodium and bacteria growth history: A preliminary study

The effect of total calcium, sodium concentration and bacteria growth history was investigated on biofilm formation of Geobacillus stearothermophilus in milk formulations. Biofilm cell numbers of three G. stearothermophilus strains were compared in two milk formulations (MF1 and MF2) over 18 h. MF1 and MF2 had similar compositions but MF2 had relatively high sodium and low calcium concentrations. Biofilm formation of all three strains was lower (P < 0.05) in MF2 than in MF1, but the inhibition of MF2 was dependent on the growth history of bacterial inocula. Supplementation of MF1 with sodium decreased (P < 0.05) biofilm formation at 18 h for dairy isolates A1 and P3. Supplementation of MF2 with 26 mm calcium increased (P < 0.05) biofilm formation of A1 at 18 h, and P3 at 10 h. The cationic profile of MF2 seemed to be important in reducing biofilms of G. stearothermophilus.

Isolation, screening, and molecular identification of new lactic acid bacteria as potential probiotics

Background and objective Lactic acid bacteria (LAB) are the most useful microorganisms to society, LAB are used in the production of many fermented food products that stimulate digestion and immunity. Therefore, the objective study aimed to isolate, characterize, and identify new LAB members and determining their potential probiotic properties. Materials and methods This study was performed through isolation, selection, and identification of LAB strains morphologically, biochemically, and genetically. Then, to determine their probiotics traits, which include acid resistance, bile-salt tolerance, β-galactosidase activity, heat sensitivity, salinity, cholesterol reduction, and antioxidant activity. Results and discussion Eight LAB isolates were isolated and purified from milk and cheeses, out of eight isolates, K2 showed the highest resistance to acidic pH 2.0, where Log CFU/ml reduced from the initial count 8.79±0.17 at pH 7.0 to 4.57±0.10 at pH 2.0 with 51.99% survival rate. Also, the six bile-tolerant isolates (K1, K2, K3, K4, A11, and A5) were found tolerant to 1% bile-salt concentrations for 3 h with 92.50, 71.10, 64.35, 60.72, 71.11, and 72.21% rate of survival, respectively. Also, antibiotic susceptibility of A11 isolate was the most sensitive, but isolate A2 was the most resistant against antibiotics. Besides, heat sensitivity of selected LAB isolates (K1, K2, A11, and B1) were able to survive at temperatures 15, 37, and 45°C, but isolates (K3, K4, A5, and A2) did not grow at 45°C. Additionally, NaCl tolerance clearly revealed that both isolates (K2 and A11) were able to tolerate a wide range of NaCl (1–8% w/v), then the growth declined with the increase of salt (10%) concentration. The results showed that the cholesterol assimilation was reduced by 32.66 and 30.20% for A11 and K1 isolates, respectively. Also, K2 isolate showed the highest antioxidant activity (86.93%), and the best β-galactosidase producer (24.65 U/ml). Therefore, the 16S-rRNA sequencing was carried out to identify four promising LAB isolates that could be employed as a probiotic strain. Phylogenetic analysis showed that isolates belonged to three genera, including Lactobacillus, Lactococcus, and Enterococcus. Strains were Lactobacillus casei (A5) KU510332, Enterococcus faecium (A11) KU510333, Lactococcus lactis (K1) KU510334, and E. faecium (K2) KU510335. Conclusion Our results presenting four new isolates as promising potential probiotics for further application in processing probiotic products.

First Report of Root Rot Caused by Calonectria montana on Sugar Beet in Heilongjiang Province, China.

Sugar beet (Beta vulgaris L.) is an important crop that has significant economic value in northern regions of China, especially in Heilongjiang Province. In October 2019, root rot was discovered on the sugar beet cultivar HDW09 in Hulan (126°64' E, 46°00' N), Heilongjiang Province, China. Typical symptoms included lesions on root tissues, which were initially small and dark brownish, then gradually turned into irregular shapes and black in color. As the disease progressed, the extent of necrosis penetrated from external layers into inner tissue. Root tissues suffered from severe decay, which resembles symptoms of several previously reported root rot diseases of sugar beet(Harveson 2006). To identify the pathogen, pieces of the transition zones (3-5mm) between asymptomatic and symptomatic tissues were surface sterilized for 15 seconds in 1% NaClO, rinsed twice with sterilized distilled water， plated on corn meal agar supplemented with penicillin G (50 mg/L), and incubated at 25 ± 2°C in the dark. Isolates belonging to a Calonectria sp. were recovered and purified using the hyphal tipping technique. Four isolates including A1, A5, A6, and A7 were used for morphological characterization and identification by DNA sequencing. The seven-day-old colonies on malt extract agar produced buff and wooly aerial mycelia. They were sienna to umber in color. Chlamydospores and microsclerotia were produced abundantly throughout the medium. For further identification, the isolates were cultured on potato dextrose agar (PDA) at room temperature(25°C) under near-ultraviolet light irradiation. Macroconidiophores comprised of a stipe, a penicillate arrangement of fertile branches, a stipe extension, and a terminal vesicle. Stipe extension were septate, straight to flexuous, 61-117 μm long, 2-4 μm wide at the apical septum, terminating in a sphaeropedunculate vesicle, 5-9 μm diam. Conidiogenous apparatus were 31-177 μm long, and 16-110 μm wide(n=30). Primary branches of conidiogenous apparatus were aseptate or 1-septate, 16-51 × 3-7 µm; secondary branches aseptate, 6-31 × 2-7 µm; tertiary branches aseptate, 8-19 × 2-6 µm, each terminal branch producing 1-6 phialides. Conidia cylindrical were rounded at both ends, straight, 32-53 × 3-5 µm, (mean = 47 × 4 µm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colorless slime(n=100). Partial sequences of calmodulin (Carbone et al. 1999), histone H3, the translation elongation factor 1-alpha, and beta-tubulin 2 (Crous et al. 2004) genes of the four isolates were obtained and deposited into GenBank under accession numbers MW118652 to MW118667. BLAST results showed that the calmodulin, histone H3, the translation elongation factor 1-alpha and beta-tubulin 2 sequences of A1, A5, A6 and A7 were highly identical to the sequences of Ca. montana strain CERC 8957 MF527082.1 (CAL) (99-100%), CERC 8930 MF527061.1 (HIS) ( 98-99%), HSP4 MN356465.1 (EF1-alpha) (100%) and HSP4 MN356460.1 (tub2) (98-99%), respectively. A phylogenetic tree using the maximum likelihood algorithm and sequences of the four concatenated genes was reconstructed in RAxML and revealed that the four isolates clustered in the clade of Ca. montana. The pathogen was identified as Ca. montana based upon these morphological and molecular traits(Liu et al. 2017; Stępniewska et al. 2020). Ten eight-week-old sugar beet plants without root rot symptoms were selected for pathogenicity test. The roots near the ground were carefully cleaned with hands. One mycelial plug (5 mm in diameter) from a seven-day-old colony of isolate A1 were used to inoculate each sugar beet root. Ten plants inoculated with plugs of noncolonized PDA served as the control plants. Pathogenicity tests were repeated three times. Plants were incubated under greenhouse conditions at 25 ± 2°C and watered when the surface soil appeared dry. All inoculated plants showed symptoms that resembled those in the field after 30 days, while the control plants remained healthy. The symptomatic tissues were plated in corn meal agar for 7 days at 25°C. Ca. montana was reisolated from 100% of the inoculated tissues, and identification was confirmed by molecular sequencing, validating Koch's postulates. This is the first report of Ca. montana in China causing root rot on sugar beet. The study suggests its broader host range and wider geographical distribution than ever known and lays a basis for further monitoring and managing this important pathogen.

A salt tolerant Sphingosinicella microcystinivorans A3 isolated from soil contaminated with mercury in traditional gold mining of Jendi Village, Wonogiri District, Indonesia

Abstract. Sutami, Purwanto, Rosariastuti R. 2021. A salt tolerant Sphingosinicella microcystinivorans A3 isolated from soil contaminated with mercury in traditional gold mining of Jendi Village, Wonogiri District, Indonesia. Biodiversitas 22: 3785-3791. Isolation and characterization of indigenous bacteria from the soil of traditional gold mining contaminated with mercury is the first step in a series of research to explore and utilize indigenous bacteria in Jendi's area. This study was aimed to determine the characteristics and identity of bacterial isolates from soil of traditional gold mining in Jendi Village, Wonogiri contaminated by mercury. The methods used in this study included bacterial isolation, media preparation, phenotypic identification including; morphological and physiological tests and genotyping tests. The results showed that the bacterial isolate A3 grew optimally in media with the addition of 10% NaCl, at a temperature of 27°C, and pH 9. There were negative reactions to the observations of gram staining, acid production from glucose, indole production, catalase and urease, and positive reactions to oxidation. A neighbor-joining phylogenetic tree based on the 16S rRNA gene sequence showed that the A3 strain was closely related to Sphingosinicella microcystinivorans strain Y2T (JCM 13185T) with 100% Query coverage and a maximum identity of 99.56%.

Analysis of the fermentative profile of yeasts isolated from natural sources

The natural environment aggregates an immense amount of microorganisms, some unknown and others still unexplored and that may have the potential for the production of compounds. Thus, this study aims to evaluate the fermentative profile of isolates from natural sources under different growing conditions. For this purpose, the plating performing on a 2% YPD solid medium incubating in an oven at 30 °C. The isolated colonies use for the carbon source assimilation test, which carried out through cell growth. Carrying out in test tubes containing the YP fermentative added with carbon sources (glucose, lactose, sucrose, fructose, mannose, maltose, starch) with a concentration of 17 ºBrix, at pH 4 and 6, incubated at 28 and 35 ºC and at different times of cultivation, aliquots removed for analysis. For the control, the yeast Catanduva-1 was used. The isolates grew in the range of pH 4 and pH 6 at 28 ºC and isolate A4 showed greater growth at a temperature of 35 °C. Isolates A4, A5, and A6 used most sugars except for lactose. Isolate A2 assimilated glucose and fructose and A6 showed a fermentation profile similar to standard yeast.

Pathological and Molecular Characterization of Magnaporthiopsis maydis Isolates Causing Late Wilt in Maize

In this study, sixteen isolates of Magnaporthiopsis maydis were isolated from infected maize (Zea mays L.) plants collected from different governorates in Egypt. These isolates were identified at the molecular level using a specific primer. All isolates have the same growth pattern form (rhizoid), growth elevation (raise) and growth margin (filiform) on PDA medium but differed in color. The faster isolate in growing on PDA medium was isolate S3 while the slower one was A2. The analysis of variance showed significant differences among isolates. The pathogenicity test was carried out under greenhouse conditions using a Single cross pioneer 3062 hybrid. Isolates F13 and F14 were highly aggressive (33.33%), while isolate Mi5 was the lowest (10%). No correlation was detected between disease incidence and radial growth of M. maydis isolates. Genetic diversity among isolates was studied using six RAPD markers and showed a high similarity percentage (95%) between isolate F13 and F14 and between isolate F14 and F15.Whereas, the lowest similarity percentage (26%) was between isolate A2 and KS11. The Cluster analysis using the Dice coefficient divided the studied isolates into five clusters. Cluster 1 contains isolates nos. F14, F15, F13, B12, KS7, and KS16, cluster 2 contains isolates nos. F9, M10, Mi5, and M8, cluster3 contains isolates nos. S3, KS6, and F1-B4, cluster 4 contains isolate number KS11 and cluster 5 contains isolate number A2.

Development of cell culture infectious clones for hepatitis C virus genotype 1b and transcription analysis of 1b-infected hepatoma cells

Globally, hepatitis C virus (HCV) genotype 1b is the most prevalent, and its infection has been found to associate with a higher risk of hepatocellular carcinoma (HCC) than other genotype viruses. However, an efficient infectious HCV genotype 1b culture system is unavailable, which has largely hampered the study of this important genotype virus. In this study, by using a systematic approach combining the sequences of infectious 1a TNcc clone and adaptive mutations, we succeeded in culture adaption of two full-length 1b clones for the reference strain Con1 and a clinical isolate A6, and designated as Con1cc and A6cc, respectively. Con1cc and A6cc replicated efficiently in hepatoma Huh7.5.1 cells, released HCV infectivity titers of 104.1 and 103.72 focus forming units per milliliter, respectively, and maintained the engineered mutations after passages. Both viruses responded to sofosbuvir and velpatasvir in a dose-dependent manner. With culture infectious 1b clones, we characterized the transcriptomes of 1b Con1cc-infected cells, in comparison with 2a-infected and uninfected cells. In conclusion, we have developed two infectious clones for genotype 1b and shown a novel strategy for culture adaptation of HCV isolates by using a genetically close backbone sequence. Furthermore, this study provides transcriptional landscape of HCV 1b-infected hepatoma cells facilitating the study of genotype 1b infection.

Development of Cell Culture Infectious Hepatitis C Virus Genotype 1b Clones and 1b-Induced Angiogenesis and Cholesterol Disorders

Globally, hepatitis C virus (HCV) genotype 1b is most prevalent, and its infection has been found to associate with a higher risk of hepatocellular carcinoma (HCC) than other genotype viruses. However, an efficient infectious HCV genotype 1b culture system is unavailable, which has largely hampered the study of this important genotype virus. In this study, by using a systematic approach combining adaptive mutations and infectious 1a TNcc sequences, we succeeded in culture adaption of two full-length 1b clones for the reference strain Con1 and a clinical isolate A6, and designated as Con1cc and A6cc, respectively. Con1cc and A6cc replicated efficiently in hepatoma Huh7.5.1 cells, released HCV infectivity titers of 104.1 and 103.72 focus forming units per milliliter, respectively, and maintained the engineered mutations after passages. Both viruses responded to sofosbuvir and velpatasvir in a dose-dependent manner. With culture infectious 1b clones, we characterized the transcriptomes of 1b Con1cc-infected cells. Con1cc infection activated angiogenesis and disturbed cholesterol homeostasis compared with uninfected cells. In conclusion, we have developed infectious clones for genotype 1b and shown a novel strategy for culture adaptation of HCV isolates by using a genetically close backbone sequence. Besides, this study provides transcriptional landscape of HCV 1b-infected hepatoma cells facilitating the study of genotype 1b infection. Funding Statement: This work was supported by the National Natural Science Foundation of China (No. 81971938 for Y.-P.L.; 81772923 for Y.Z.), The Innovation Research Team for Basic and Clinical Studies on Chronic Liver Diseases of 2018 High-Level Health Teams of Zhuhai (for Y.-P.L. and F.X.), National Key Basic Research Program of China (No. 2015CB554301 for Y.-P.L.), Entrepreneurial Talent Team Award of Guangdong Province (No. 2016ZT06S252 for Y.-P.L.). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The use of patient serum was approved by the Medical Ethics Committee at the Zhongshan School of Medicine, Sun Yat-sen University (No. 2014-072).

Isolation, identification and potential probiotic characterization of lactic acid bacteria from Thai traditional fermented food.

The probiotic potential of lactic acid bacteria (LAB) isolated from Thai traditional fermented food was investigated. Forty-two samples were collected from four markets in Phra Nakhon Si Ayutthaya Province. Out of 50 isolated LAB, 6 (a3, f4, f8, K1, K4 and K9) obtained from pla-ra and bamboo shoot pickle samples showed high tolerance to gastrointestinal tract conditions. These isolates were selected to identify and characterize their probiotic properties. Isolate a3 was identified as Weissella thailandensis, isolates f4 and f8 were identified as belonging to Enterococcus thailandicus and isolates K1, K4 and K9 were determined as Limosilactobacillus fermentum. All six LAB exhibited high autoaggregation ability (93.40–95.01%), while W. thailandensis isolate a3 showed potential for coaggregation in almost all the pathogenic bacteria tested. Cell-free supernatant (CFS) obtained from all isolates did not inhibit Staphylococcus aureus. CFS derived from L. fermentum isolate K4 showed the most efficient antimicrobial activity, in particular against Gram-negative bacteria, while L. fermentum isolate K4 presented high surface hydrophobicity in the presence of xylene and n-hexane. All LAB isolates were found to be resistant to clindamycin and nalidixic acid, whereas E. thailandicus isolate f8 exhibited resistance to most of the antibiotics tested. L. fermentum isolate K4 showed promise as a suitable probiotic candidate for future applications in the food industry due to tolerance to gastrointestinal tract conditions with high surface hydrophobicity and inhibited most of the pathogens tested.

Isolation, Characterization and Activity Test of Soil Origin Bacteria Amilage

This study aimed to obtain amylase-producing potent bacteria from soil and test the amylase activity produced. Soil samples were taken from the Biological Education and Research Forest, Andalas University. The isolation was done by using the stratified dilution technique on agar media. The screening of amylase activity employed the qualitative and quantitative tests on agar starch. From 8 isolated amylolytic bacteria, there were three isolates with amylolytic potential. The results of characterization and identification based on Bergey's Manual of Determinative Bacteriology show that isolates A4, A1, and A6 belonged to the genus Bacillus, Corynebacterium, and Klebsiella. The bacteria obtained can then be produced and optimized for the needs of industrial enzymes.

Isolation of Bacillus spp. from rhizosphere of garden soil: their potential role in amylase production and nitrogen cycle

Rhizobacteria influence plant growth by producing various substances like enzymes and play role in nitrogen cycle. Microbes are the most important sources of enzymes because of their stability and reduced price in production. In our present investigation, soil samples were collected from rhizosphere of garden soil and a total of eight isolates of Bacillus spp. were presumptively identified by morphological, cultural and biochemical analysis with the conventional technique. The isolates were named as A1, A3, A4, A5, B2, C1, D1 and D2. Amylase enzyme assay was performed by 3, 5-dinitrosalicylic acid method. The highest enzyme activity was observed with the isolate A4 (2.71 U/ml) followed by isolates D2 (2.54 U/ml) and D1 (2.52 U/ml). The highest ammonification was found to be positive with the isolates A4 and B2 followed by isolate D1. Denitrification potential was found to be highest with isolates A4 and A5. No isolates were found to fix atmospheric nitrogen in Jensen’s media after two weeks of incubation. The isolates have a great potential of amylase production and they can be used in different industries as well as in starch rich waste degradation. Involvement of these bacteria in nitrogen cycle may help promote growth of plants.&#x0D; Stamford Journal of Microbiology, Vol.10 (1) 2020: 12-15