Isolates N1 Research Articles

ANTIOXIDANT ACTIVITY OF TEMBESU (Fagraea fragrans Roxb.) LEAF

Herbal medicine is becoming a trend in curing several diseases especially degenerative because of the bad effects of synthetic drugs. Tembesu plant (Fagraea fragrans Roxb.) is traditionally used for degenerative diseases. This study aims to separate bioactive compounds (active fraction), pure isolate compounds from tembesu leaf, then observe antioxidant activity. The methods used are maceration extraction, liquid-liquid fractionation, purification of compounds by gravity chromatography column and antioxidant activity by scavenging DPPH radicals. The results showed that the active antioxidant fractions were n-hexane and ethyl acetate fractions. The results of purification of antioxidant compounds, obtained 6 isolates N1, N2, N4, N5, E8 and E11. Antioxidant activity (IC50) of pure isolates N1 (13.72 ppm), N2 (28.93 ppm), N4 (110.44 ppm), N5 (177.23 ppm), E8 (82.50 ppm) and E11 (12 ,86 ppm). The antioxidant compounds that have been isolated are terpenes (N1, N2, N4, E8), phenols (N5) and flavonoids (E11). This study concluded that tembesu leaf have antioxidant potential. Antioxidant bioactive compounds in tembesu leaf have the potential to be developed into standardized herbs and phytopharmaceuticals.

Antibacterial Activity of Essential Oil from Rosemytle Leaves (Rhodomyrtus tomentosa (Ait.) Hassk)

Rosemytle leaves (Rhodomyrtus tomentosa (Ait.) Hassk) have been used by society to treat various diseases related to bacterial infections, such as dysentery and typhoid fever caused by Shigella dysenteriae and Salmonella typhi, respectively. This study aimed to evaluate the antibacterial activity of essential oils from rosemytle leaves against both bacterias. Extraction was performed with a macerating device using n-hexane, ethyl acetate, and ethanol extracts, sequentially. This study used the agar diffusion method to test the antibacterial activity applied to the essential oils with concentrations of 1000, 500, 250, 125, 62.5, and 31.25 μg/mL. The antibacterial test results showed that n-hexane and ethyl acetate extracts were active against both bacterias while ethanol extract was not. Then, isolates N1 and E1 were produced respectively from n-hexane extract and ethyl acetate extract. The MIC values of both N1 and E1 for S. dysenteriae, and S. typhi were the same, namely 125 μg/mL. Isolate N1 was an essential oil containing menthol (59.60%), caryophyllene (25.77%), and cubenol (14.63%) while isolate E1 was an essential oil containing (73.93%), pentanone (8.30%), alpha calacorene (7.58%), and calacorene (3.78%). Rosemytle leaves have the potential to be developed as a drug to treat dysentery and typhoid fever.

Isolation of calcite precipitation bacteria to improve the strength of concrete

Concretes are the second most consumed material on earth. However, it is susceptible to micro crack formation and has pores in it. The repairing cracks in concrete require high cost and labor and traditional repair system are chemical based, expensive and lead to environmental and health hazards. In this study, the calcite precipitation bacteria were investigated. Bacterial strains were isolated from cement samples and were tested for urease activities, potential to form endospore and calcite precipitation. The results showed that four candidates were isolated with high urease activities and calcite precipitation. Among them, the bacterial isolate N1 could precipitate 0.5 g CaCO3 per liter. By using 16S rDNA sequencing, this strain was identified as Bacillus thuringiensis. The ability of this strain to tolerate the extreme environment of cement, it could be used in remediating the cracks and fissures in various building or concrete structure.

Identification of epiphytic yeasts and bacteria with potential for biocontrol of grey mold disease on table grapes caused by Botrytis cinerea

The objective of this study was to identify grapevine epiphytic yeasts and bacteria for biocontrol of Botrytis cinerea on grapes. Antagonistic yeasts and bacteria were isolated from the epiphytic flora associated with grape berries and leaves cv. ‘Thompson seedless’ from vineyards in Iran and identified by sequencing the conserved genomic regions. A total of 130 yeast and bacterial isolates from the surface of grapevine were screened in vitro for determining their antagonistic effect against B. cinerea and used to control postharvest gray mold. Among the 130 isolates, five yeasts and four bacterial isolates showed the greatest antagonistic activity in vitro against B. cinerea. Two yeasts species including Meyerozyma guilliermondii and Candida membranifaciens had high antagonistic capability against the pathogen. Also, 4 bacterial isolates belonging to Bacillus sp. and Ralstonia sp. showed significant biocontrol effect against B. cinerea. The isolates were capable of producing volatile and non-volatile substances, which suppressed the pathogen growth. The antagonistic activity of selected yeasts and bacteria against the pathogen was investigated on wounded berries of ‘Thompson seedless’. On small clusters with intact berries, all of the antagonistic isolates considerably reduced the decay on grape berries and inhibition of gray mold incidence on fruits treated by these isolates was less than 50%, except for the isolate N1, which had higher capability in inhibiting the disease incidence. These results suggest that antagonist yeasts and bacteria with potential to control B. cinerea on grape can be found in the microflora of grape berries and leaves.

Isolation and characterization of marine bacteria from macroalgae Gracilaria salicornia and Gelidium latifolium on agarolitic activity for bioethanol production

Gracilaria salicornia and Gelidium latifolium have high content of agar and potential to be use as raw material for bioethanol. In bioethanol production, one of the processes level is enzyme hydrolysis. Various microorganisms, one of which is bacteria, can carry out the enzyme hydrolysis. Bacteria that degrade the cell walls of macroalgae and produce an agarase enzyme called agarolytic bacteria. The purpose of this study was to isolate bacteria from macroalgae G. salicornia and G. latifolium, which has the highest agarase enzyme activities, and to obtain agarase enzyme characteristic for bioethanol production. There are two isolates bacteria resulted from G. salicornia that are N1 and N3 and there are two isolates from G. latifolium that are BSUC2 and BSUC4. The result of agarase enzyme qualitative test showed that isolates bacteria from G. latifolium were greater than G. salicornia. The highest agarolitic index of bacteria from G. salicornia produced by isolate N3 was 2.32 mm and isolate N3 was 2.27 mm. Bacteria from G. latifolium produced by isolate BSUC4 was 4.28 mm and isolate BSUC2 was 4.18 mm, respectively. Agarase enzyme activities from isolates N1 and N3 were optimum working at pH 7 and temperature 30 °C, while from isolates BSUC4 was optimum at pH 7 and temperature 50 °C. This is indicated that the four bacteria are appropriate to hydrolyze macro alga for bioethanol production.

Evaluation of Resistance to Phytophthora sojae in Soybean Mini Core Collections Using an Improved Assay System.

Stem and root rot disease caused by Phytophthora sojae is devastating to soybean crops worldwide. Developing host resistance to P. sojae, considered the most effective and stable means to control this disease, is partly hampered by limited germplasm resources. In this study, we first modified conventional methods for a P. sojae resistance assay to a simpler and more cost-effective version, in which the P. sojae inoculum was mixed into the soil and the resistance was evaluated by survival rate (%) of soybean seedlings. This rating had significant correlations (P < 0.01) with the reduction in root fresh weight and the visual root rot severity. Applying this method to evaluate P. sojae resistance in soybean mini core collections comprising either 79 accessions originating from Japan (JMC) or 80 accessions collected around the world (WMC) revealed a wide variation in resistance among the individual varieties. In total, 38 accessions from the JMC and 41 from the WMC exhibited resistance or moderate resistance to P. sojae isolate N1 (with virulence to Rps1b, 3c, 4, 5, and 6), with ≥50% survival. Of these, 26 from the JMC and 29 from the WMC showed at least moderate resistance to P. sojae isolate HR1 (vir Rps1a-c, 1k, 2, 3a-c, 4-6, and 8). Additionally, 24 WCS accessions, in contrast to only 6 from the JMC, exhibited 100% survival after being challenged with both the N1 and HR1 isolates, suggesting a biogeographical difference between the two collections. We further verified two JMC varieties, Daizu and Amagi zairai 90D, for their resistance to an additional four P. sojae isolates (60 to 100% survival), which may provide new and valuable genetic sources for P. sojae resistance breeding in soybean.

Virus symptomatology in accessions of the Medicago truncatula core collection and identification of virus resistance phenotypes

Plants of 212 accessions from the core collection of model legume species Medicago truncatula were inoculated with infective sap containing Alfalfa mosaic virus (AMV, isolate EW), Bean yellow mosaic virus (BYMV, isolate MI) or Cucumber mosaic virus (CMV, isolate SN-1). A diverse range of systemic symptoms were obtained that varied widely in severity depending on the combination of virus isolate and accession, or, especially with AMV, some accessions became infected but did not display symptoms. The delay between virus inoculation and symptom appearance normally varied from 1 to 4 weeks, but with CMV it took up to 8 weeks in two accessions. Five (AMV), 59 (BYMV) and 22 (CMV) core accessions remained uninfected systemically. Plants of most of these accessions, and some that died or gave susceptible phenotypes, were then inoculated with two additional isolates of AMV (eight accessions), or two distinct strains of BYMV (58 accessions) and CMV (21 accessions). Plants of accession 11715 remained uninfected by CMV isolates CP (CMV subgroup 1) and LW (CMV subgroup 2), but those of all other previously uninfected accessions became infected systemically by all three viruses. All accessions inoculated with AMV isolates Aq and Hu, and most inoculated with BYMV isolate LKoj1-NN (generalist strain), BYMV isolate LP-1 (lupin strain), and CMV isolates CP and LW developed typical susceptible phenotypes. However, systemic hypersensitive phenotypes developed with BYMV LKoj1-NN and LP-1 in plants of 4456, or with LKoj1-NN only in 774, 1526, 4327, 14829, 15268, 22922 and 25654; 15268 and 25654 had developed this phenotype previously with MI (generalist strain). Similarly, plants of 21362 developed this phenotype with CMV CP and LW, while plants of 1526, 2748 and 31443 developed it with CP; 2748, 21632 and 31443 had developed it previously with SN-1 (mixture of subgroups 1 and 2). Once the genetic bases of the BYMV and CMV resistances found in M. truncatula are understood, they may prove useful in future virus resistance breeding among crop and pasture legumes.

English

The D2/D3 LSU rDNA expansion segment of 13 isolates attaching tea shrubs roots in tea gardens that verified by morphological and morphometrical studies as Pratylenchus loosi Loof, 1960 from Guilan province, North of Iran, were amplified and sequenced. Amplification of the D2/D3 LSU rDNA expansion segments yielded one fragment at over all sequenced isolates as 787 bp in size. The DNA sequences were aligned using Clustral X1.81 together and with three sequences of similar region of P. loosi isolates available in Genbank database (Isolate T from Serilanka and Isolates N1 and N2 from Florida, USA). Also the genetic distance between sequences data were calculated through four methods as following; Uncorrected distance (UC), Jukes-Cantor (JC) Kimura distance (K) and Jin-Neigamma distance (JNG). For generating phyllogenetic trees both Neighbor-joining (NJ) and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) were used. The results indicated that very short genetic distance exist among the Iranian isolates and between the Iranian isolates and isolate T from Serilanka whereas the Iranian isolates and isolate T were genetically distinct from isolates N1 and N2. The phyllogenetic analyses revealed relationship not only among Iranian isolates but also between Iranian isolates and isolate T.

Effect of natural sequence variation at the H-2Ld-restricted CD8+ T cell epitope of the murine cytomegalovirus ie1-encoded pp89 on T cell recognition.

The amino acid sequence YPHFMPTNL of pp89, the ie1-encoded product of murine cytomegalovirus (MCMV; Smith strain), constitutes an immunodominant T cell epitope recognized in association with H-2Ld. Nucleotide sequencing of MCMV isolates derived from wild mice identified variation between amino acids 147-192 of pp89 in 19 of 27 isolates, including the region encompassing the CTL epitope (amino acid residues 168-176). Four groups of isolates with naturally occurring variant sequences for the CTL epitope were defined: (1) YPHFMPPNL; (2) YPHFMPPSL; (3) YPHFIPPSL; and (4) YLDFMPPNL. The remaining isolates, and the laboratory strains K181 and Vancouver, showed complete identity with the Smith strain. Polyclonal pp89 (Smith strain)-specific CTL only weakly recognized target cells infected with MCMV from most variant groups. No lysis of cells infected with isolate N1 from group 4 was detected. Analyses of cross-reactive recognition of YPHFMPTNL peptide-coated targets by CTL primed with variant MCMV isolates showed that the group 2 and 3 isolates, G4 and K6, respectively, but not the group 4 isolate N1, elicited CTL that exhibited a cross-reactive response. Furthermore, while the group 2 and 3 isolates G4 and K6 were able to prime CTL responses that displayed reactivity to homologous pp89 variant nonapeptides, the group 4 isolate N1 failed to do so. Finally, while immunization of mice with the nonapeptide YPHFMPTNL conferred significant protection against the laboratory strain K181 [correction of Kl81], no evidence of protection was observed for the group 2 and 4 variants G4 and N1, respectively. These observations raise the possibility that clinical isolates of HCMV may also differ in sequence from potential vaccine strains at immunodominant epitopes for CD8+ T cells thus reducing the efficacy of vaccination.

Proteolytic activity of rumen fungi belonging to the genera Neocallimastix and Piromyces

Proteolytic activity of two rumen fungal isolates Neocallimastix sp. strain N1 and Piromyces sp. strain P1 was examined. Proteases are active between pH 6.5 and 9.0 with maximum at 7.9 for isolate N1 and between 6.5 and 10.5 with maximum at 8.8 for isolate P1. Proteolytic activity increased as temperature increased until 50°C and a sudden decrease at 60°C was observed in both isolates. EDTA, 1,10-phenanthroline, p-chloromercuribenzoate (PCMB), merthiolate and phenylmethylsulphonyl fluoride (PMSF) were effective proteolytic inhibitors against both isolates.

The virobacterial agglutination test as a rapid means of detecting cocoa swollen shoot virus

SummaryThe virobacterial agglutination (VBA) test was developed as a means of detection of cocoa swollen shoot virus (CSSV). Identification of CSSV‐infected Theobroma cacao in the field has only been possible by visual examination of symptoms, by virus transmission using mealybugs and by grafting to induce symptom expression in Amelonado cocoa seedlings. Detection of latent infection has not been possible even using enzyme‐linked immunosorbent assays (ELISA). The VBA test successfully detected CSSV in infected sap diluted to 1/2560. Antisera to a range of mild and severe CSSV isolates were tested, and the results suggest a close relationship between seven isolates (1A, Bosomtwi, Bosomuoso, Nkrankwanta, Nsaba, Seidi‐Nkawie and SS365B) while the mild isolate N1 appears to be less closely related. The VBA test was compared with both direct and indirect ELISA in the field. Only VBA detected all the cocoa trees which were known to be infected and additionally identified infection in many symp‐tomless trees.