Surface Sterilization Method Research Articles

Isolasi Fungi Endofit Daun Kopi Robusta (Coffea canephora)

Endophytic fungi are organisms that live in plant tissues that have the potential to produce the same secondary metabolites as their host. This study attempted to isolate and also characterize endophytic fungi from Robusta coffee leaves (Coffea canephora). Data collection was carried out in several stages, namely isolation of endophytic fungi by surface sterilization method, and conventional characterization of endophytic fungi by observing the morphology of endophytic fungi. The results showed that as many as 4 isolates of endophytic fungi were isolated from Robusta coffee leaves (Coffea canephora) with macroscopic characteristics of white color with orange circle edges, cotton-like shape, and green and orange spots on the underside of the fungus.

Flower endophytic fungi of Geodorum densiflorum endangered orchid

Geodorum densiflorum is an ornamental and medicinally important orchid. The medicinal metabolites are produced by endophytic fungi associated with orchid tissue, particularly flowers. However, there is no report of the endophytic fungi from the G. densiflorum flowers. This research aimed to study the endophytic fungi from different parts of G. densiflorum flower. Fungal isolation was carried out from sepal, petal, labium, stigma, and anther using surface sterilization method. The fungi were identified by combining morphological and molecular characteristics of ITS rDNA region. The results showed that each flower organ had different species of endophytic fungi. A total of seven species of endophytic fungi were obtained; four species were successfully identified by molecular identification and three species based on morphology. The four species, namely Hypomontagnella barbarensis, Aspergillus oryzae, Curvularia pseudobranchyspora, and Nigrospora chinensis, while the three species, namely Gonatobotrys sp., Humicola sp., Aspergillus section Nigri. The labium inhabited by Curvularia pseudobranchyspora, Nigrospora chinensis, Aspergillus section Nigri. Three species isolated from petals, namely Hypomontagnella barbarensis, Gonatobotrys sp., and Aspergillus oryzae. The sepal is inhabited by Humicola sp. There were no endophytic fungi in stigma and anther. This indicates that each flower part is a unique habitat of endophytic fungi.

Optimization of cultural conditions affecting improved bioactive metabolite production by endophytic fungus Trichoderma harzianum

The present study aimed to optimize cultural conditions for optimum bioactive metabolite production by endophytic fungus Trichoderma harzianum, isolated by surface sterilization method from the leaf of the eucalyptus plant. The fungus was identified based on morphological characterization. Fungal metabolites were carried out by ethyl acetate solvent. The antibacterial activity was tested against Escherichia coli (ATCC 25922) and Staphylococcus aureus (NCTC 6571). Various carbon, nitrogen sources, pH, temperature, incubation period, and NaCl on the antibacterial metabolite production were studied. Bioactive metabolite production of T. harzianum exhibits a broad spectrum of in vitro antibacterial activity against two strains of bacteria. For the optimum production of bioactive metabolites, Dextrose and Glucose were found to be the best sources of carbon and the best sources of Nitrogen Yeast extract (YE) and (NH4)2SO. The maximum production of bioactive metabolites occurs at pH 7 and 25°C.; the NaCl showed a positive influence on bioactive metabolites.

Application of Barley Tweaky Spike Mutants for the Study of Effects of Plant Immunity-Related Substances

Barley developmental mutants tweaky spike (tw) with disturbed auxin pathways possess a unique feature of an increased level of mouldy germinating grains (MGG), which serves as a convenient model to investigate the effects of plant immunity-related substances. The effects of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D), auxin inhibitors, salicylic acid (SA), and trans-cinnamic acid (TCA) were studied using the tw-WT system in surface-sterilized and unsterilized germinating grains under high rates of natural infection. Significant differences among the allelic tw mutants were revealed at the natural MGG level and in response to 2,4-D, SA, and TCA. The most effective means against MGG were sterilization and TCA. 2,4-D inhibited root growth in tw and tw2 mutants, occurring only in unsterilized and not sterilized germinating grains, while the opposite was observed for TCA and SA. The tw mutations influenced variations in the seed-borne fungal spectra, decreasing the frequency of Bipolaris sorokiniana and increasing Fusarium spp. Hypochlorite-based surface sterilization methods should be used with caution in studies where the action of exogenous 2,4-D will be analysed in germinating grains. Auxin pathway disturbances specific for pleiotropic tw mutants are generally restricted to organogenesis but not to germination events.

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds.

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

Indigenous fungi from corn as a potential plant growth promoter and its role in Fusarium verticillioides suppression on corn

Indigenous fungi can suppress infection by pathogens and produce secondary metabolites that directly or indirectly affect plant growth. This study aimed to test indigenous fungi collected from corn plants as biological control agents and their effects on the viability and vigor of corn seeds. Purposive sampling method was used for sampling where soil samples taken from the rhizosphere zone, corn stem and leaf tissue from three locations namely Maros-South Sulawesi, Bone-South Sulawesi, Sigi-Central Sulawesi, Indonesia. Rhizospheric fungi were isolated from soil collected at the rhizosphere and rhizoplane using a serial dilution technique, while the endophytic fungi isolated from the leaves and stem tissues using surface sterilization method. The isolated fungi were cultured on a potato dextrose agar (PDA) medium. An antagonism test was performed using the dual culture method on PDA media with F. verticillioides as target pathogen. Pathogenicity test and the effect of fungi on corn seed germination was carried out using the blotter test method. Parameters observed were; necrotic symptoms on seedlings, growth potential, germination, growth rate, growth simultaneity, vigor index, germination rate, and time needed for 50% of the total germination. The effect of the isolated indigenous fungi on corn growth was carried out in-planta using seedling trays. The results of the blotter test and in-planta test were further confirmed by a physiological characteristic test. And assessing the fungi's ability to dissolve potassium, phosphate, and produce protease enzymes. A total of 89 fungal isolates were isolated and collected from various parts of the corn plant. Nineteen of the 89 fungal isolates showed inhibitory activity against F. verticillioides by ≥ 50% inhibition. The fungal isolates JRP 5 MRS, JRP 9 MRS, JRP 10 MRS, JRP 7 MRS, and JEDF 1B BN were selected based on the tests and showed a consistently positive effect on seed viability and vigor with a value of ≥90%. The isolates did not cause necrosis in corn, and had the ability to suppress the growth of pathogenic F. verticillioides by ≥ 50%.

Culturable Fungal Community of Pterocladiella capillacea in Keelung, Taiwan: Effects of Surface Sterilization Method and Isolation Medium.

Fungi associated with macroalgae are less known when compared with those on wood in the marine environment. In this study, we assessed the diversity of fungi associated with the red alga Pterocladiella capillacea at Chao-Jin Park, Keelung, Taiwan. Algal segments of healthy and dead thalli were washed/sterilized with different solutions (sterile artificial seawater, 70% ethanol, and 4% sodium hypochlorite), plated on three different media (glucose-yeast extract-peptone seawater agar (GYPS), potato dextrose seawater agar (PDAS), and artificial seawater agar (SA)), and isolated as pure cultures. Identification was mainly based on BLAST search analysis of the internal transcribed spacers of rDNA (ITS). The highest isolation frequency (no. of segment with fungi/total no. of segment × 100) was in dead thalli (61.23%), thalli washed with seawater (88.38%), and thalli plated on GYPS (62.10%). A total of 3187 isolates were cultured, representing 129 taxa (in 67 genera); the higher species richness was isolated from healthy thalli (119 species), thalli washed with seawater (111 species), and on GYPS (112 species). Ascomycota (Eurotiales, Hypocreales, Capnodiales, Pleosporales, Xylariales) dominated the fungal community in P. capillacea with many basidiomycetous yeasts and few Mucoromycota. Aspergillus, Cladosporium, Penicillium (Ascomycota), and Rhodosporidium (Basidiomycota) were the dominant genera associated with the alga. The surface washing/sterilization schemes of algal thalli affected fungal diversity, but the isolation media used did not. While these genera are known producers of antimicrobial secondary metabolites, they might form a mutualistic relationship with P. capillacea by exchanging nutrients from photosynthesis for protection from microbial diseases.

Isolation, antibacterial activity, and molecular identification of endophytic fungi from Pogostemon cablin

Pogostemon cablin is the medicinal plants that produces patchouli essential oils as secondary metabolites which has multiple functions including antibacterial ability. The secondary metabolites in plants mostly associated with their endophytic fungi. In this study we isolated endophytic fungi from Pogostemon cablin’s leaves and examined antibacterial activity of the endophytic fungi against Escherichia coli and Staphylococcus aureus, as well as find out the identity of most potential isolate based on Internal Transcribe Spacer (ITS) region. The isolation of endophytic fungi was performed using surface sterilization method on Malt Extract Agar (MEA) medium. The antibacterial activity was tested using paper disc on Muller Hinton Agar (MHA) medium and molecular identification was amplified using ITS 4 and ITS 5 primers. The isolation process resulted in 5 isolates of endophytic fungi. The antibacterial assay indicated one potential isolate with the highest antibacterial activity when tested against E. coli and S. Aureus, exhibited 20.9 mm and 19 mm clear zone respectively. Molecular identification from ITS region database depicted that the potential isolate has high homology with Nigrospora sp. by 99% similarity. This result suggested that the antibacterial ability of essential oils from the Pogostemon cablin’s leaves might has high correlation with the occurrence of endophytic fungi.

Anti-bacterial effect of marine sea grasses mediated endophytic actinomycetes against K. pneumoniae

In this study, the pure, pale white, powdery nature and pigment produced endophytic actinomycetes strains were isolated from marine Sea grasses sample. The isolated strains were endophytes as confirmed by surface sterilization and validation methods. The anti-bacterial efficacy materials of the crude compound were extracted by liquid–liquid extraction using various organic solvents. Anti-bacterial efficacy of the isolated actinomycetes strain RMN 10 was exhibited excellent anti-bacterial activity against K. pneumoniae at increasing concentration. In addition, the minimum inhibition concentration (MIC) of the crude actinomycetes compound has excellent anti-bacterial activity at 250 µg/mL concentration and this concentration was fixed as a MIC. Further, the structural and shape damages of actinomycetes crude compound treated K. pneumoniae cells were clearly identified in SEM images. All the invitro experiments were proved that the actinomycetes compound has excellent anti-bacterial activity against K. pneumoniae.

Plant tissue culture of Nicotiana tabacum cv. TAPM 26 and its minimum inhibition against herbicide-Dalapon

Current study is to establish a basic plant tissue culture of Nicotiana tabacum TAPM 26 and test the plant tissue on resistancies against 2,2 DCP an active ingredient in herbicide-Dalapon. During micropropagation, the surface sterilization method was ascertained on seeds of tobacco. HgCl2 was used to disinfect tobacco seeds at different concentrations (0.05 gL-1, 0.2 gL-1, 0.5 gL-1 and 1.0 gL-1) within three minutes. About 70% seeds were survived when exposed to 0.05 gL-1 of HgCl2, whereas, no seeds were germinated when sterilized at concentrations above 0.05 gL-1 of HgCl2. To optimize an efficient protocol of shoots and callus formation during in vitro regeneration, explant types and plant growth were studied. Growth regulators NAA (0.1 mgL-1, 0.2 mgL-1, 0.5 mgL-1, 1.0 mgL-1 and 2.0 mgL-1) and BAP (1.0 mgL-1, 2.0 mgL-1, 3.0 mgL-1 and 4.0 mgL-1) were used. The explants types were one month old leaves and two weeks old cotyledons. The maximum numbers of shoots per explants were obtained from cotyledon with combination 0.1 mgL-1 NAA and 1.0 mgL-1 BAP. The highest callus fresh weight was achieved when NAA 0.5 mgL-1 with BAP 1.0 mgL-1 after four weeks. Thus, the highest number of shoots produced per explants from leaves culture on the MS media containing 0.2 mgL-1 NAA and 4.0 mgL-1 BAP. The best callus fresh weight was obtained with combination of 1.0 mgL-1 NAA and 1.0 mgL-1 BAP by using leaves explant. Finally, Dalapon (5 gL-1, 10 gL-1, 15 gL-1 and 20 gL-1) were applied onto leaves and cotyledon cultures of N. tabacum to check on the minimum concentration of inhibition. The minimum concentration of inhibition of leaves and cotyledon cultures of N. tabacum was at 5 gL-1 of 2,2DCP but not at 10 gL-1, 15 gL-1 and 20 gL-1. This investigation will shed alight for future studies on transgenic tobacco resistant against Dalapon

Microbially-mediated carbonate dissolution and precipitation; towards a protocol for ex-situ, cave-analogue cultivation experiments

Subterranean calcite dissolution and precipitation are often considered as strictly geochemical processes. The active involvement of microbes in these processes is commonly underestimated in the literature due to general oligotrophic conditions in caves, except in particular cave conditions, such as sulfidic caves and moonmilk deposits, where the presence of microbes likely plays a key role in mineral deposition. Here, we study the possible involvement of microbes from Postojna Cave, Slovenia, in carbonate dissolution (litholysis) and precipitation (lithogenesis). Microbes were sampled from small pools below hydrologically diverse drip sites and incubated on polished limestone tablets at 10 and 20°C for 2 and 14 weeks under cave-analogue conditions. The tablets were then observed under scanning electron microscope to investigate microbe–rock interactions. Our experiments showed the presence of various microbial morphotypes, often associated with extracellular polymeric substances, firmly attached on the surfaces. Unfortunately, our surface sterilization method using 96% and 70% ethanol could not establish the complete aseptic conditions in deep natural cracks in the experimental limestone tablets. Nonetheless, our results emulate the interaction of environmental microbes with limestone rock. Conspicuous calcite dissolution and precipitation were observed in association with these microbes. Furthermore, we show evidence of entombment of microbes in a Si-rich precipitate during nutrient-depleted growth conditions and we suggest that microbial involvement in silica mobilization under ambient conditions may be a widespread and often overlooked phenomenon. Our findings have important implications for microbial-mediation of cave carbonate dissolution and precipitation, including the preservation of past climate proxy signals in speleothems and prehistoric cave art. Improvements to the methodology and further work are suggested to enable more robust ex-situ cultivation experiments in the future, facilitating better and more detailed research into this topic.

Study on Endophytic Fungi Associated with Moringa oleifera Lam. Collected from Lombok Island, West Nusa Tenggara

An assemblage of endophytic fungi was isolated from Moringa oleifera Lam. collected from Lombok island, West Nusa Tenggara Province, Indonesia. Fungal endophytes were isolated using surface sterilization methods with slight modification. Forty-six selected endophytic fungal strains were isolated from the leaves, petioles, and stems of M. oleifera. The fungal strains identification through morphological observation and ITS rDNA-based molecular analysis showed that fungal endophytes were associated with host plants belonging to the taxa Alternaria, Cladosporium, Colletotrichum (Glomerella), Corynespora, Curvularia (Cochliobolus), Fusarium, Mucor, Ochrocladosporium, Phomopsis (Diaporthe), and Trametes. In this study, endophytic Phomopsis dominating the obtained strains, whereas 26% (12/46) strains were isolated from host plant samples.

Time-Modulated LF-Microwave Hybrid Plasma for Surface Sterilization

The plasma surface sterilization method has emerged as a new sterilization method for medical instruments with many advantages, including nontoxicity, short treatment time, and low thermal damage to the sterilized material, over conventional sterilization methods. In this study, low-frequency (LF)-microwave hybrid plasma is produced by irradiation of microwave to an LF plasma jet. This hybrid plasma is characterized by atmospheric-pressure nonthermal plasma with a large amount of reactive species and has been applied for plasma surface sterilization. To reduce the treatment temperature while maintaining the effect of surface sterilization for applicability to heat-sensitive materials, the LF-microwave hybrid plasma was modulated in time by using pulsed microwaves. It was found that the gas temperature of the LF-microwave hybrid plasma produced by pulsed microwaves can be much lower than that of the LF-microwave hybrid plasma produced by continuous microwaves. Moreover, the gas temperature of the plasma was found to depend on the pulsewidth and duty ratio of microwaves used to produce it even if the time-averaged microwave power remained constant. Analyses of the optical emission spectra and chemical indicator tests indicated that the number of reactive oxygen radicals produced in the LF-microwave hybrid plasma also depends on the pulsewidth and duty ratio of the microwaves. In addition, it was found that the dependence of the number of oxygen atoms produced in the LF-microwave hybrid plasma on the microwave pulsewidth is opposite to that of the gas temperature. Spore-forming bacteria ( Geobacillus stearothermophilus ) with a population of $2.4 \times 106$ CFU/ $0.25{''} \times 1.5$ ” in a paper strip were sterilized for 1 min at a treatment temperature of 82.43 °C using pulsed LF-microwave hybrid plasma. The temperature and time of sterilization with the pulsed LF-microwave hybrid plasma were found to be lower than those with continuous LF-microwave hybrid plasma.

A New RoVe Method to Purify Endophytic Fungi from Medicinal Plants Western Ghats of Karnataka India

Endophytic fungi from medicinal plants were hardly ever reported as compared to those from soil and marine sources. The present results associate the presence of endophytic fungi in medicinal plants by isolating them from three medicinal plants i.e. Bombax ceiba, Aloe vera, and Ximenia americana. In the present research investigation, surface sterilization method and media were standardized, and 32 endophytic fungi were isolated from three medicinal plants. We assessed the competence of three different surface sterilization methods and four media for isolation of endophytic fungi. The RoVe method used was more effective in eliminating epiphytic microorganisms. Therefore by using new method we have isolated total of 32 fungal endophytes and those were belongs to Aspergillus nomius (63.20%), Aspergillus niger (41.60%), Thielaviopsis basicola (38.33%), Fusarium oxysporum (33.20%), Pestalotiopsis inflexa (27.20%), Nigrospora sphaerica (20%), Alternaria alternata (15.30%) and Phomopsis archeri (20%). This is the first report of successful isolation of endophytic fungi from the said medicinal plants, for using newly formulated surface sterilization method.

Isolation and identification of flavonoid-producing endophytic fungi from medicinal plant Conyza blinii H.Lév that exhibit higher antioxidant and antibacterial activities

BackgroundConyza blinii H. Lév is a medicinal plant that has a variety of pharmacological activities, but its study is at a standstill due to the shortage of resources.MethodThis study utilized the surface sterilization method to isolate endophytic fungi, and they were preliminarily identified by morphology. Flavonoid-producing strains were screened by NaNO2-Al(NO)3 colorimetry and further identified by the ITS sequence. Additionally, we used five antioxidant assays (DPPH, Hydroxyl radical, ABTS, FRAP and T-AOC assays) to systematically evaluate the antioxidant capacity of total flavonoids , and we also determined their antibacterial activity.ResultsIn this study, 21 endophytic fungi were isolated from wild Conyza blinii H. Lév for the first time. There were six flavonoid-producing strains, especially CBL11, whose total flavonoid content reached 50.78 ± 2.4 mg/L. CBL12, CBL12-2 and CBL1-1 all exhibited excellent antioxidant activity. The effect of CBL12 was similar to that of ascorbic acid at low concentrations, and its radical scavenging rates for DPPH and ABTS were 94.56 ± 0.29 % and 99.88 ± 0.27%, respectively, while its IC50 values were only 0.11 ± 0.01 mg/mL and 0.2 ± 0.01 mg/mL. Through LC-MS, we found that CBL12 could produce many high-value flavonoids, such as 3-methoxyflavone, nobiletin, formononetin, scopoletin, and daidzein. Additionally, CBL9 had good antibacterial activity against both gram-positive and gram-negative bacteria. Notably, we obtained the high-yield strains CBL12 and CBL9, which not only had high yields (10.64 ± 1.01 mg/L and 10.17 ± 0.11 mg/L, respectively) but also had excellent biological activity. Hence, the results of this study provide new ideas for endophytic fungi that can be exploited as a source of flavonoids and other medicinal components from Conyza blinii H. Lév. Moreover, this study can serve as a reference for the development of rare medicinal materials.

An assemblages of fungal endophytes isolated from medicinal plants collected from Toba and Samosir, North Sumatra

Endophytic fungi are a group of microorganisms that grow in plant tissue without causing symptoms of disease in host plants. Fungal endophytes have a great diversity and potential as an agent of bioactive compound and metabolites that are important in the pharmaceutical industry, agriculture, and environment. In this study isolation and identification of fungal endophytes inhabiting an assemblages of medicinal plants collected from Toba and Samosir Regency, North Sumatra, Indonesia were carried out. Fungal endophytes were isolated using surface sterilization methods with slightly modification and totally 88 selected endophytic fungal strains were isolated from roots, stems, barks, leaves, and fruits of 12 species of medicinal plants. The fungal strains identification through morphological observation showed that selected fungal endophytes were associated with host plant belonging to the taxa Colletotrichum, Fusarium, Lasiodiplodia, Neopestalotiopsis, Phoma, Phomopsis, Phyllosticta, Schizophyllum, Trichoderma, and Xylaria. In this study endophytic Phomopsis dominating the obtained strains where 30% (27/88) strains of them were isolated from 8 species of medicinal host plants. Preliminary exploration of fungal endophytes through isolation and identification was the first step in order to discover the potential strains were able to produce promising metabolites and bioactive materials. The results of the study are expected to be initial microbial resources and information for screening and utilization of endophytic fungi through bioprospecting.

Optimization of in vitro asymbiotic seed germination protocol for Serapias vomeracea

Abstract Serapias vomeracea is an economically important orchid species which is over-collected from nature, because of its glucomannan-rich tubers. Thus, optimization of in vitro culture methodology in this species is required to meet industrial needs and to secure its populations in nature. This study aimed to optimize the surface sterilization protocol for S. vomeracea seeds and to select the optimal seed germination medium by comparing the commonly used media in in vitro orchid culture. During seed surface sterilization, ethyl alcohol (EtOH) pre-treatment prior to sodium hypochlorite (NaOCl) treatment increased the disinfection success and viable seed yield when examined using the triphenyl tetrazolium chloride (TTC) seed viability test. Also, low-g force centrifugation as an additional step in the surface sterilization method separated the seeds without embryo from the viable seeds and thereby decreased potential counting errors after incubation. Comparison of media showed that solid Knudson C (KN) medium induced the highest number of germinated seeds. However, seed germination success of Lindemann (LN) and Vacin & Went (VW) media was found to be higher when the media was used in liquid form. Half-strength liquid VW was the only medium that induced higher germination success than the other full-strength media. The highest number of ungerminated seeds was found when using KN medium whereas liquid VW medium gave the lowest number. In general, protocorm formation was triggered when the media were used in liquid form. However, rhizoid elongation was suppressed in liquid media. These findings suggest that this optimized seed surface sterilization method offers a simple and effective alternative to classical methods. Additionally, solid KN medium may be considered as a cost-effective and reliable alternative to other commonly-used complex media in S. vomeracea cultures.

Dryas as a Model for Studying the Root Symbioses of the Rosaceae.

The nitrogen-fixing root nodule symbiosis is restricted to four plant orders: Fabales (legumes), Fagales, Cucurbitales and Rosales (Elaeagnaceae, Rhamnaceae, and Rosaceae). Interestingly all of the Rosaceae genera confirmed to contain nodulating species (i.e., Cercocarpus, Chamaebatia, Dryas, and Purshia) belong to a single subfamily, the Dryadoideae. The Dryas genus is particularly interesting from an evolutionary perspective because it contains closely related nodulating (Dryas drummondii) and non-nodulating species (Dryas octopetala). The close phylogenetic relationship between these two species makes Dryas an ideal model genus to study the genetic basis of nodulation by whole genome comparison and classical genetics. Therefore, we established methods for plant cultivation, transformation and DNA extraction for these species. We optimized seed surface sterilization and germination methods and tested growth protocols ranging from pots and Petri dishes to a hydroponic system. Transgenic hairy roots were obtained by adapting Agrobacterium rhizogenes-based transformation protocols for Dryas species. We compared several DNA extraction protocols for their suitability for subsequent molecular biological analysis. Using CTAB extraction, reproducible PCRs could be performed, but CsCl gradient purification was essential to obtain DNA in sufficient purity for high quality de novo genome sequencing of both Dryas species. Altogether, we established a basic toolkit for the culture, transient transformation and genetic analysis of Dryas sp.

Surface sterilization methods impact measures of internal microbial diversity in ticks

BackgroundTicks are obligate blood feeders transmitting major pathogens worldwide. Over the past few years, considerable research efforts have focused on the diversity, distribution and impact of gut and intracellular bacterial symbionts on tick development and tick-borne pathogen transmission. The study of this internal microbiome requires the use of a sterilization method to remove external (i.e. cuticular) microbes present on the tick’s surface and to avoid any further contamination. Several sterilization methods exist, including ethanol- or bleach-based treatments that are both effective in killing microbes but with different potential effects on DNA denaturation.MethodsWe examined how these different sterilization methods impact the measure of internal microbial diversity hosted by the Cayenne tick Amblyomma cajennense (sensu stricto). Bacterial barcoding investigations based on 16S rRNA gene sequences were conducted on two batches of 50 individuals each: Ticks of the first batch were sterilized with bleach diluted at 1% and the second batch with 70% ethanol. Tick external microbiome was also determined from cuticle smearing and water samples used for tick washing.ResultsBacterial barcoding investigations showed major differences between ethanol- and bleach-treated specimens. Both methods led to the detection of major intracellular bacteria associated with A. cajennense (s.s.) but ethanol-treated ticks always harbored a higher bacterial diversity than bleach-treated ticks. Further examinations of tick gut and tick external microbiome revealed that ethanol-based surface sterilization method is inefficient to eliminate the DNA of external bacteria.ConclusionsWe herein provide evidence that studies investigating the internal microbiome of ticks should consider bleach as the gold standard to efficiently remove cuticular bacterial DNA. Indeed, this method does not impact the internal bacterial diversity hosted by ticks and is thus a better method than the ethanol-based one for studying the internal microbiome.

Keragaman Bakteri Endofit Penghasil L-Asparaginase Bebas L-Glutaminase

Endophytes are potential as a source of active compound producer. Endophytes that is isolated from tropical medicinal plants has been getting attention due to its high biodiversity and active compound producing ability. L-asparaginase is the first enzyme used as chemotherapeutic agent for leukemia. The aim of this study is to collect the endophytic bacteria associated with tropical medicinal plants from Sumba Island and investigate the activity of L-asparaginase without L-glutaminase from endophytic bacteria isolates. The samples Gliricidia sepium, Pittosporum moluccanum, Clerodendrum buchanani, and Zingiberaceae are collected from Wanggameti, East Sumba, NTT. Samples are sterilized using surface-sterilization method and endophytic bacteria are isolated using plant piece method on R2A media. Selected endophytic bacteria are identified by 16S rDNA sequences. L-asparaginase screening is conducted using modified R2A with addition of L-asparagine and phenol red as colour indicator. A total of 34 isolates of endophytic bacteria were collected from 5 samples. A total of 14 genus consisted of 17 different bacterial species were obtained from 34 selected isolates. Endophytic bacteria of P. stutzeri strains of SMKL1 and R. radiobacter strains of SMKW2 from the Kahili plant were needed as L-glutaminase-free L-asparaginase and were potential to be candidate of leukemia cancer chemotherapy