Universal Primer Set Research Articles

Abundance and community composition of comammox bacteria in different ecosystems by a universal primer set

Complete ammonia oxidizing bacteria (CAOB) have been recognized as a new member of ammonia-oxidizing microorganisms (AOMs) due to its single-step nitrification capability. However, the abundance and diversity of CAOB in environmental ecosystems were still far from known owing to the lack of specific molecular marker. Herein, a universal primer set specifically targeting both clades of CAOB amoA gene with high specificity and coverage was successfully designed. Intriguingly, real-time quantitative PCR tests revealed that CAOB were ubiquitous and unexpectedly abundant in agricultural soils, river sediments, intertidal zones, drinking water and wastewater treatment systems. Phylogenetic analysis indicated that clade A existed in all the five types of ecosystems, while clade B were only detected in soil and sediment samples. Four sub-clusters were further classified within clade A, in which N. nitrosa cluster dominated CAOB amoA in activated sludge samples while the new recognized soil cluster was the primary constitute in soils. Moreover, the niche specialization between different CAOB species and the environmental conditions were supposed to be the primary driven force to shape the diversity and community of CAOB. This study provided a strong evidence in support of the ubiquities and high abundances of CAOB in various environmental ecosystems and highlighted the significance of including CAOB as the new member of AOMs to re-evaluate the biogeochemical nitrogen cycle.

Development and Evaluation of Isothermal Amplification Methods for Rapid Detection of Lethal Amanita Species.

In the present work, loop-mediated isothermal amplification (LAMP) and hyperbranched rolling circle amplification (HRCA) methods were developed to detect and distinguish different lethal Amanita species. Specific LAMP primers and HRCA padlock probes for species-specific identification and a set of universal LAMP primers for lethal Amanita species were designed and tested. The results indicated that the LAMP-based assay was able to discriminate introclade lethal Amanita species but was not able to discriminate intraclade species perfectly, while the HRCA-based assay could discriminate whether introclade or intraclade species. The universal LAMP primers were positive for 10 lethal species of Amanita section Phalloideae and negative for 16 species of Amanita outside section Phalloideae. The detection limits of LMAP and HRCA were 10 and 1 pg of genomic DNA per reaction, respectively. In conclusion, the two methods could be rapid, specific, sensitive and low-cost tools for the identification of lethal Amanita species.

Host DNA integrity within blood meals of hematophagous larval gnathiid isopods (Crustacea, Isopoda, Gnathiidae)

BackgroundJuvenile gnathiid isopods are common ectoparasites of marine fishes. Each of the three juvenile stages briefly attach to a host to obtain a blood meal but spend most of their time living in the substrate, thus making it difficult to determine patterns of host exploitation. Sequencing of host blood meals from wild-caught specimens is a promising tool to determine host identity. Although established protocols for this approach exist, certain challenges must be overcome when samples are subjected to typical field conditions that may contribute to DNA degradation. The goal of this study was to address a key methodological issue associated with molecular-based host identification from free-living, blood-engorged gnathiid isopods—the degradation of host DNA within blood meals. Here we have assessed the length of time host DNA within gnathiid blood meals can remain viable for positive host identification.MethodsJuvenile gnathiids were allowed to feed on fish of known species and subsets were preserved at 4-h intervals over 24 h and then every 24 h up to 5 days post-feeding. Host DNA extracted from gnathiid blood meals was sequenced to validate the integrity of host DNA at each time interval. DNA was also extracted from blood meals of wild-fed gnathiids for comparison. Attempts were also made to extract host DNA from metamorphosed juveniles.ResultsUsing a cox1 universal fish primer set, known fish host DNA sequences were successfully identified for nearly 100% of third-stage juvenile gnathiid blood meals, digested for up to 5 days post-feeding. For second-stage juveniles, host identification was 100% successful when gnathiids were preserved within 24 h of collection. Fish hosts were positively identified for 69% of sequences from wild-fed gnathiid isopods. Of the 31% of sequences not receiving a ≥ 98 % match to a sequence in GenBank, 25 sequences were of possible invertebrate origin.ConclusionsTo our knowledge, this is the first study to examine the degradation rate of gnathiid isopod blood meals. Determining the rate at which gnathiids digest their blood meal is an important step in ensuring the successful host identification by DNA-based methods in large field studies.

Development of the cephalopod-specific universal primer set and its application for the metabarcoding analysis of planktonic cephalopods in Korean waters.

Although spatiotemporal analysis of the cephalopod larvae provides the useful information for the effective management of their resources, it has been difficult mainly due to their low numbers in the mixed zooplankton net samples and difficulty in morphological identification. In order to analyze the planktonic cephalopods using next-generation sequencing (NGS), we have designed a cephalopod-specific universal (CPD) primer set targeting a region covering mitochondrial cytochrome b and ND6 genes based on the currently identified 36 complete cephalopod mitochondrial genome sequences in the GenBank database. The expected amplicon sizes by CPD primers were between 465 and 471 bp, which was applicable to the MiSeq system (Illumina, San Diego, CA, USA). NGS results of pooled DNAs from 8 months (including 739 zooplankton net samples) collected from Korean waters in 2016 showed the exclusive cephalopod sequences with little contaminant sequences supporting the specificity of CPD primer set. Total 47 representative cephalopod haplotypes (seven families and 10 genera) were obtained from 1,439,414 merged reads. Among the total analyzed haplotypes, Watasenia scintillans, Todarodes pacificus, and Sepiola birostrata were the most abundant species in Korean waters. Two “unidentified” clades in order Oegopsida were identified, which was showed less than 90% sequence identity but closely related to Enoploteuthidae and Idiosepiidae, respectively. Monthly changes in proportions of each haplotype were also identified, which may reflect its reproduction and spawning period. The larvae of W. scintillans was dominant from February to June, while high proportions of other cephalopod taxa were also identified from August to November. Only single haplotype was dominant in W. scintillans (Type 2) throughout the year, while two distinct haplotypes showed seasonal differences in T. pacificus.

First Report of Lasiodiplodia theobromae and Neofusicoccum parvum/N. ribis species Complex Causing Fruit Rot on Strawberry in the United States

During the last three seasons, approximately 1% of strawberry (Fragaria × ananassa) with symptoms of fruit softening and discoloration were observed in three fields in Florida. Fruit eventually became black and mummified with pycnidia embedded in the tissue that produced hyaline, aseptate conidia. Eleven isolates recovered from infected tissue were purified by growing them on 2% water agar (WA) with sterilized pine needles (Pavlic et al. 2004) and transferring a single conidium to a new 2% WA plate, by spreading a conidial suspension over the medium and selecting a single germinating conidium 16 h later. DNA of the 11 isolates was extracted from 2-week-old colonies and PCR was performed following the conditions of White et al. (1990) and Carbone and Kohn (1999). Two sets of universal primers, ITS1F/ITS4 and EF1-728F/EF1-986R, were used to amplify and sequence the internal transcriber spacer (ITS) and partial translation elongation factor 1-alpha (EF1-α) regions, respectively. Sequences compared with the GenBank database using BLASTn showed 99 to 100% identity with ITS and EF1-α sequences within the Neofusicoccum parvum/N. ribis species complex or Lasiodiplodia theobromae: N. parvum (MH057203 and KM921768; n = 2), N. kwambonambiense (n = 3), N. ribis (n = 1), N. brasiliense (n = 2), and L. theobromae (MH049696 and KX580762; n = 3). All sequences were deposited in GenBank with following accession numbers: N. parvum/N. ribis species complex (MH809520 to MH809527 for ITS, and MH809508 to MH809515 for EF1-α), and L. theobromae (MH809517 to MH809519 for ITS, and MH809505 to MH809507 for EF1-α). Mycelia of both groups on V8 agar medium were initially white and aerial, but Lasiodiplodia mycelia became light gray after 3 to 5 days and Neofusicoccum mycelia turned dark gray to olivaceous. Conidia of the Neofusicoccum group were aseptate, hyaline, oblong, and 15.0 to 22.9 × 6.1 to 10.4 μm (n = 25). Lasiodiplodia conidia were similar but larger, 22.6 to 33.0 × 13.0 to 19.4 μm (young conidia) and when mature, became brown, one septate, with longitudinal striations, measuring 25.9 to 34.8 × 14.8 to 19.6 μm (n = 25). Similar morphology was previously reported (Alves et al. 2008; Pavlic et al. 2009). To confirm pathogenicity, four isolates (two from N. parvum/N. ribis species complex, and two L. theobromae) were selected and incubated for 2 weeks at 23°C under continuous light until formation of pycnidia. Twenty mature strawberry fruit of ‘Florida Radiance’ were inoculated in the field (five fruit per isolate), by wounding the fruit with a forceps and placing a single pycnidium inside the wound. Field conditions during the incubation period ranged from 16 to 29°C and 65 to 80% relative humidity. Disease incidence was assessed 6 to 10 days after inoculation, and the experiment was repeated once. After 10 days, fruit inoculated with the N. parvum/N. ribis were completely dark and covered with pycnidia, whereas fruit inoculated with L. theobromae had 80 to 100% of its surface covered with pycnidia. All 20 inoculated fruit showed the same symptoms as described above. Both fungi were reisolated from inoculated fruit, fulfilling Koch’s postulates. N. parvum and N. kwambonambiense have been previously reported to cause a postharvest disease of strawberry in Brazil (Lopes et al. 2014). To our knowledge, this is the first report of N. parvum/N. ribis species complex and L. theobromae causing fruit rot in strawberry fields in the U.S. This study sheds light on the possible broad distribution of these pathogens on different hosts as N. parvum was recently reported in the same area causing shoot blight and stem canker on pomegranate (KC and Vallad 2016).

Detection of potyviruses infecting Iranian common beans using broad-spectrum antibodies and universal primer pairs

Six provinces of Iran were surveyed during 2012–2014 to detect potyviruses infecting common bean crops. A total of 277 leaf samples were collected from bean plants and some other legumes as well as weeds and wild legumes showing virus-like symptoms. Enzyme-linked immunosorbent assay (ELISA) using broad-spectrum potyvirus antibodies gave a positive reaction with 63 samples (22.7%). Moreover, a mixed colony of aphids (Aphis craccivora and A. fabae), collected from a locust tree plant (Robinia sp.), transmitted a potyvirus to young healthy bean plants; the infection was confirmed through serological assays. Partial nuclear inclusion b (NIb) and coat protein (CP) regions of the genomes of 19 ELISA-positive samples were amplified by reverse-transcription polymerase chain reaction (RT-PCR) using NIb and NWCIEN (or WCIEN) universal primer sets, respectively. Potyvirus species identification was confirmed by BLASTN and phylogenetic analyses followed by ELISA tests using virus-specific antibodies or biological assays. The results clearly showed the presence of Bean common mosaic virus (BCMV), Bean common mosaic necrosis virus (BCMNV), Bean yellow mosaic virus (BYMV) and Wisteria vein mosaic virus (WVMV) in the samples tested. Non-specific amplification of a Cucumovirus genome by using NIb primers was also recorded. The results showed the natural occurrence of a new potyvirus species (WVMV), and a new host for BYMV (Trifolium repens) in the mid-Eurasian region of Iran. Our findings indicate the capacity of the universal detection systems for fast and accurate detection of legume potyviruses in the country and for use in routine diagnostic assays.

Profiling the Clostridia with butyrate-producing potential in the mud of Chinese liquor fermentation cellar

Butyrate and its derivates pertain to the key aroma contributors of strong-flavour baijiu, a kind of Chinese liquors, that is produced from grains by solid-state multispecies anaerobic fermentation in a mud cellar. Microbes inhabiting in the fermentation pit mud largely determines baijiu's flavour and quality. In order to shed light on the microbial functional groups driving butyrate production in pit mud, clone library analysis was firstly performed and the results demonstrated that Clostridia (relative abundance: 50%) and Bacilli (37%) were major groups possessing butyrate kinase (buk) pathway and Clostridia (98%) dominated butyryl-CoA:acetate CoA-transferase (but) pathway. According to Clostridial specific-16S rRNA gene sequencing analysis, we found the resilience character of Clostridial community in pit mud. Amongst Clostridial groups, 32.0% of the sequences were grouped into Clostridiales incertae sedis, followed by Heliobacteriaceae (18.3%) and Clostridiaceae 1 (8.4%). Moreover, Hydrogenispora, Sedimentibacter and Clostridium were the top three abundant genera. Relative abundance of Hydrogenispora was higher in the late days of fermentation, while Sedimentibacter exhibited higher proportion in the early days. Different from the previous studies using universal bacterial primer sets, Hydrogenispora was first reported as one dominant genus in pit mud. As for the reported potential butyrate producer Clostridium, nineteen species were obtained and ten of them were first isolated from the pit mud. Amongst them, buk was identified in eleven species by PCR analysis, while but was identified in the other seven, indicating the species-specific butyrate synthesis pathways of Clostridium. This study provides a perspective on targeting and isolating specific functional microbes in baijiu microbiota with the gene sequence-based medium prediction method.

First Report of Pectobacterium carotovorum subsp. brasiliense Causing Blackleg and Stem Rot Disease of Potato in Russia

Blackleg and stem rot of potato (Solanum tuberosum) were observed in several commercial fields in the central part of Russia with up to 40% incidence during 2012 to 2017. Symptoms included decay of internal root and stem tissues, blackening of petiole vascular bundles, and partial yellowing of leaves. The disease can cause serious potato yield losses in the field and storage in Russia. Pectobacterium carotovorum subsp. carotovorum, P. atrosepticum, Dickeya dianthicola, and D. solani are the main causal agents of blackleg disease in Russia (Kornev et al. 2012). Surveys conducted in commercial fields produced multiple samples showing stem rot symptoms. Bacteria were isolated from symptomatic tissues by suspending 1-cm stem sections with lesions in sterile tap water for 10 min, followed by serial dilutions on crystal violet pectate agar (CVP). Bacterial colonies producing pitting on CVP were reisolated and purified on nutrient broth yeast extract medium. Characterization of three isolates provided evidence that a previously unreported blackleg/stem rot pathogen was present in the surveyed region. F126 (from potato ‘Lady Clare’, Samara Region), F128 (from potato ‘Sante’ from Kaluga Region), and F140 (from Moscow region) were gram negative, facultatively anaerobic, and able to grow at 37°C on nutrient agar. They produced typical stem rot symptoms in potato plants inoculated with 10⁷ CFU/ml of the pathogen by stem injection and incubated at 25°C for 48 h. The strains were reisolated successfully from symptomatic potato to complete Koch’s postulates. These strains were negative in polymerase chain reaction (PCR) analyses using specific primers Y45/Y46 for the detection of P. atrosepticum and ADE1/ADE2 for Dickeya (Czajkowski 2011). In contrast, all strains yielded an amplicon (322 bp) in a PCR with primers Br1f and L1r for P. carotovorum subsp. brasiliense (Duarte et al. 2004). 16S rRNA was amplified using universal primer set F27/R1492 (Monciardini et al. 2002). The strains F126, F128, and F140 (GenBank accessions MG570140, MG570141, and MG570142) had 99% sequence identify with 16S rRNA of P. carotovorum subsp. brasiliense type strain BC1 (CP009769.1). Additional loci representing two housekeeping genes were amplified and sequenced: gyrB (MG570137, MG570138, and MG570139) and recA (MG570134, MG570135, MG570136). Primer sets used were as reported by Young and Park (2007) and Dauga (2002). All strains showed 97 to 98% similarity with type strain P. carotovorum subsp. brasiliense BC1. To our knowledge, this is the first report of blackleg and stem rot caused by P. carotovorum subsp. brasiliense on potato in the Russian Federation. According to the data of several diagnostic laboratories, P. carotovorum subsp. brasiliense occurred in >20% potato seed stocks harvested in 2017 and was the predominant blackleg pathogen in fields in the Moscow region in 2018.

The design and testing of mini-barcode markers in marine lobsters.

Full-length mitochondrial cytochrome c oxidase I (COI) sequence information from lobster phyllosoma larvae can be difficult to obtain when DNA is degraded or fragmented. Primers that amplify smaller fragments are also more useful in metabarcoding studies. In this study, we developed and tested a method to design a taxon-specific mini-barcode primer set for marine lobsters. The shortest, most informative portion of the COI gene region was identified in silico, and a DNA barcode gap analysis was performed to assess its reliability as species diagnostic marker. Primers were designed, and cross-species amplification success was tested on DNA extracted from a taxonomic range of spiny-, clawed-, slipper- and blind lobsters. The mini-barcode primers successfully amplified both adult and phyllosoma COI fragments, and were able to successfully delimit all species analyzed. Previously published universal primer sets were also tested and sometimes failed to amplify COI from phyllosoma samples. The newly designed taxon-specific mini-barcode primers will increase the success rate of species identification in bulk environmental samples and add to the growing DNA metabarcoding toolkit.

PCR-based species identification applied in Japanese pear orchards to survey seasonal proportion changes of phytoseiid mite species

For this study, we developed a PCR-based method to identify the ribosomal internal transcribed spacer (ITS) sequences of six phytoseiid mite species found in Japanese pear orchards: Neoseiulus californicus (McGregor), Neoseiulus womersleyi (Schicha), Neoseiulus makuwa (Ehara), Amblyseius eharai Amitai and Swirski, Gynaeseius liturivorus (Ehara), and Amblyseius swirskii Athias-Henriot. Using the method, we can show seasonal proportion changes of phytoseiid mite species composition in Japanese pear orchards. Genomic DNA extracted individually from phytoseiid mites collected in the orchards are subjected to PCR of the ITS sequences using a universal primer set of which nucleotide sequences are conserved among the six phytoseiid mite species. Then, DNA samples showing PCR products with the universal primer set are used for another PCR of the ITS sequences using species-specific primer sets designed for respective phytoseiid mite species. Species-specific PCR is conducted in order of decreasing dominance of phytoseiid mite species. The method, which specifically identifies the six phytoseiid mite species irrespective of their sexes and developmental stages, might be useful for researchers who are lacking adequate morphological identification skills and nucleotide sequencing systems at their institutions.

Potentially pathogenic genera of free-living amoebae coexisting in a thermal spring

Little is known about the prevalence of Balamuthia mandrillaris within the environment due to its difficult isolation, but once an axenic culture is established, it is relatively easy to maintain. As most of the time researchers are interested mainly in isolating B. mandrillaris from environmental samples, the flora that accompanies it becomes second in importance. Therefore, this study aimed to determine which potentially pathogenic free-living amoebae, in addition to B. mandrillaris, could be found co-inhabiting a source of natural thermal water called “Agua Caliente” (Mexico), where this amoeba has previously been detected twice by molecular methods. A third sampling from this same source was carried out to try to isolate B. mandrillaris and other free-living amoebae using 37 and 45 °C as isolation temperatures. For PCR techniques, specific primers were used for B. mandrillaris, Naegleria fowleri, and Acanthamoeba species, plus a universal primer set for the eukaryotic 18S SSU rRNA gene for other isolated amoebae. PCR products were sequenced for final identification. 42 strains of the primary isolate were obtained, but only 34 could be kept in culture. Of them, 23 strains were identified as Naegleria lovaniensis, eight strains as Acanthamoeba jacobsi, two strains as Stenamoeba sp. and only one was identified as Vermamoeba vermiformis. The isolation of B. mandrillaris was once again not successful, but the presence of potentially pathogenic and nonpathogenic free-living amoebae is reported for the first time in this type of water in Mexico thanks to molecular methodology.

Detection of bacterial contamination in platelet concentrates from Brazilian donors by molecular amplification of the ribosomal 16S gene.

The aim of our work was to establish a semi-automated high-throughput DNA amplification method for the universal screening of bacteria in platelet concentrates (PCs). Among cases of transfusion transmission of infectious agents, bacterial contamination ranks first in the number of events, morbidity and mortality. Transmission occurs mainly by transfused PCs. Automated culture is adopted by some blood banks for screening of bacterial contamination, but this procedure is expensive and has a relatively long turnaround time. PCs were spiked with suspensions of five different bacterial species in a final concentration of 1 and 10 colony-forming units (CFU) per millilitre. After incubation, the presence of bacteria was investigated by real-time polymerase chain reaction (PCR) and by the Enhanced Bacterial Detection System (eBDS, Pall) assay as a reference method. Real-time PCR amplification was performed with a set of universal primers and probes targeting the 16S rRNA gene. Co-amplification of human mitochondrial DNA served as an internal control. Using the real-time PCR method, it was possible to detect the presence of all bacterial species tested with an initial concentration of 10CFU mL-1 24 h after contamination, except for Staphylococcus hominis. The PCR assay also detected, at 24 h, the presence of Serratia marcescens and Enterobacter cloacae with an initial concentration of 1CFU mL-1 . The real-time PCR assay may be a reliable alternative to conventional culture methods in the screening of bacterial contamination of PCs, enabling bacterial detection even with a low initial concentration of microorganisms.

PO-080 Prevalence of human papilloma virus and its phylogenetic analysis in patients with head and neck squamous cell carcinoma in pakistan

IntroductionHNSCCs are characterised by distinct phenotypic, aetiological, biological and clinical heterogeneity. It is a heterogeneous disease that can be roughly divided into human papillomavirus (HPV) positive and negative. HPV is a recently identified causative agent for a subset of head and neck cancers, primarily in the oropharynx but very little is known about the prevalence of specific HPV types in Pakistan.Material and methodsThis cross sectional study was carried out in the Department of Immunology, University of Health Sciences, Lahore, Pakistan after approval from the Ethical Review Committee and Advanced Studies and Research Board, UHS Lahore. After an informed consent, tissue sections from a total of 72 patients with HNSCC were collected. The diagnosis of HNSCC was confirmed again on H and E staining and DNA was extracted from formalin fixed paraffin embedded tissue sections using DNA extraction kit. For the detection of HPV DNA, two universal primer sets GP5/GP6 and MY9/MY11 were used in the polymerase chain reaction. The samples, found to be positive for HPV on PCR, were sequenced for the phylogenetic analysis of HPV.Results and discussionsAmong 72 cases of HNSCC, only 5 (14.4%) cases were found to be positive for HPV by using universal primers sets. The prevalence of HPV in HNSCC in many international studies from Europe and USA have documented a prevalence of 5%–50% in HNSCC cases. After confirming the presence of HPV in HNSCC cases, positive DNA samples have been transported to a well-known international laboratory for gene sequencing and molecular diagnosis. As soon as possible, we will receive the sequencing data, we will sketch a phylogenetic tree and further subtyping of HPV and the further results will be presented.ConclusionThe findings of current study will be helpful in launching public health awareness and future vaccination programs against specific HPV types in Pakistan to lessen the burden of HPV related HNSCC.

Genomic analysis of red-tide water bloomed with Heterosigma akashiwo in Geoje.

Microbial community structures of harmful algal bloom (HAB) caused by Heterosigma akashiwo in Geoje were analyzed using the MiSeq platform. To analyze phytoplankton communities without cross-reactivity with predominant bacteria, a new phytoplankton-specific 23S universal primer set was designed by modifying two previously used ones. The new universal primer set turned out to be a useful tool for the analysis of the phytoplankton community; it showed a high specificity for phytoplankton without cross-reactivity to bacterial sequences as well as the wide taxon coverage presenting from prokaryotic cyanobacteria to eukaryotic algae. Next Generation Sequencing (NGS) data generated by two universal primer sets (16S and 23S) provided useful information about the H. akashiwo bloom. According to the 23S universal primer set, proportions of H. akashiwo increased by more than 200-fold as the bloom occurred and its numbers were high enough to detect in control sites. Its operational taxonomic units (OTUs) were detected in the bloom sites at low proportions suggesting that the 16S universal primer set may not be as effective for monitoring harmful algal blooming (HAB) as the 23S universal primer set. In addition, several abundant OTUs in Chlorophyta were not presented by the 16S universal primer set in this study. However, the 16S primer set was useful for detecting decreases in Foraminifera as HAB occurred suggesting that genomic analyses using two universal primer sets would provide more reliable data for understanding microbial community changes by various environmental or ecological events, including HAB. Genomic analyses using two universal primer sets was also useful for determining a correlation between microbial components as HAB occurred. Heterosigma akashiwo was positively correlated with other bloom species, including Karenia mikimotoi, Teleaulax amphioxeia, and bacteria in Verrucomicrobia.

Influence of Biostimulation Treatment Using Composted Plant Biomass on Bacterial Diversity of an Aged Petroleum Contaminated Soil as Determined by Culture-dependent and 16S rRNA Gene PCR-DGGE Based Identification Methods

Influence of biostimulation treatment using composted plant biomass on bacterial diversity of an aged crude oil contaminated soil (ACOCS) was determined using culture-dependent and 16S rRNA gene PCR-DGGE based identification methods. Seven treatment plots were designed and included treatments A (TPA) through G (TPG). Samples were collected bi-weekly from 7 treatment plots designed in situ during a 70-day study period that spanned 10 weeks. Composted (2,500 g each) Water hyacinth (EC), Mexican sunflower (TD) and Bermuda grass (CD) were used as nutrient supplements in 4,000 g of ACOCS in situ. TPA was un-amended while TPB, TPC, and TPD had EC, TD and CD added singly. TPE had EC and TD while TPF contained EC and CD in combination. TPG consisted of EC, TD and CD combined. Bacterial isolates were obtained on mineral salts medium and identified based on their morphological and biochemical characteristics. DGGE fingerprints of PCR-amplified 16S rRNA bacterial gene fragments were also determined using the universal primer set: 7F: 5’-GAGTTTGATCCTGGCTCAG-3’ and 1492R:5’-GGTTACCTTGTTACGACT-3’that corresponded to positions 968 and 1401 of Escherichia coli 16S rRNA gene sequence. DGGE bands fell into corresponding operational taxonomic units based on a threshold of 91-100% sequence similarity. Dendrogram showed dominant DGGE bands in TPB though TPG over time with TPA having no band. The isolates are known crude oil utilizers and are closely related to Gordonia sp. BS261404 with 98% sequence similarity, Aquitalea magnusonii KG26145 (96%), Sphingobacterium sp. K1261411 (97%) and Achromobacter sp. HQ261417 (100%). Data indicated that pseudomonads are the dominant bacteria involved in hydrocarbon biodegradation after biostimulation with the composted plant materials. Cultural and molecular methods of cultivation of microorganisms are neither contradictory nor excluding and should be considered as complementary to interrogate the bacterial diversity in the natural soil environment.

Demonstration of the potential of environmental DNA as a tool for the detection of avian species

Birds play unique functional roles in the maintenance of ecosystems, such as pollination and seed dispersal, and thus monitoring bird species diversity is a first step towards avoiding undesirable consequences of anthropogenic impacts on bird communities. In the present study, we hypothesized that birds, regardless of their main habitats, must have frequent contact with water and that tissues that contain their DNA that persists in the environment (environmental DNA; eDNA) could be used to detect the presence of avian species. To this end, we applied a set of universal PCR primers (MiBird, a modified version of fish/mammal universal primers) for metabarcoding avian eDNA. We confirmed the versatility of MiBird primers by performing in silico analyses and by amplifying DNAs extracted from bird tissues. Analyses of water samples from zoo cages of birds with known species composition suggested that the use of MiBird primers combined with Illumina MiSeq could successfully detect avian species from water samples. Additionally, analysis of water samples collected from a natural pond detected five avian species common to the sampling areas. The present findings suggest that avian eDNA metabarcoding would be a complementary detection/identification tool in cases where visual census of bird species is difficult.

Identification of ‘Candidatus Phytoplasma asteris’ causing sesame phyllody disease and its natural weed host in Jammu, India

During May–June 2016, little leaf and phyllody symptoms were observed on 8% of sesame crop in Chakbhalwal and Kotbhalwal regions of Jammu, India. Phytoplasma suspected witches’ broom symptoms on Cannabis sativa subsp. sativa was also observed in and around sesame fields with the incidence varied from 5 to 12% in different fields. Total genomic DNA from both symptomatic sesame and Cannabis plants were subjected to nested PCR assays using two sets of universal phytoplasma specific primers P1/P7 followed by R16F2n/R16R2 and 3Far/3Rev to amplify the 16S rDNA fragments. First round PCR amplification with primer pair P1/P7 did not yield expected 1.8 kb products of 16S rRNA gene region from any of the symptomatic test samples. However, DNA specific fragments of approximately 1.2 and 1.3 kb were amplified from sesame and C. sativa sub sp. sativa by using R16F2n/R16R2 and 3Far/3Rev primer pairs, respectively. BLASTn comparison and phylogenetic analysis of 16SrDNA phytoplasma sequences of sesame and cannabis phytoplasma strains revealed the association of ‘C. Phytoplasma asteris’ (16Sr I group) with symptomatic samples. The virtual RFLP analysis of the 16Sr DNA sequence of sesame and cannabis further identified as phytoplasma strains of 16SrI-B subgroup.

Novel multiplex PCR reveals multiple trypanosomatid species infecting North American bumble bees (Hymenoptera: Apidae: Bombus).

Crithidia bombi and Crithidia expoeki (Trypanosomatidae) are common parasites of bumble bees (Bombus spp.). Crithidia bombi was described in the 1980s, and C. expoeki was recently discovered using molecular tools. Both species have cosmopolitan distributions among their bumble bee hosts, but there have been few bumble bee studies that have identified infections to species since the original description of C. expoeki in 2010. Morphological identification of species is difficult due to variability within each stage of their complex lifecycles, although they can be easily differentiated through DNA sequencing. However, DNA sequencing can be expensive, particularly with many samples to diagnose. In order to reliably and inexpensively distinguish Crithidia species for a large-scale survey, we developed a multiplex PCR protocol using species-specific primers with a universal trypanosomatid primer set to detect unexpected relatives. We applied this method to 356 trypanosomatid-positive bumble bees from North America as a first-look at the distribution and host range of each parasite in the region. Crithidia bombi was more common (90.2%) than C. expoeki (21.3%), with most C. expoeki-positive samples existing as co-infections with C. bombi (13.8%). This two-step detection method also revealed that 2.2% samples were positive for trypanosmatids that were neither C. bombi nor C. expoeki. Sequencing revealed that two individuals were positive for C. mellificae, one for Lotmaria passim, and three for two unclassified trypanosomatids. This two-step method is effective in diagnosing known bumble bee infecting Crithidia species, and allowing for the discovery of unknown potential symbionts.

Awards of JSE Outstanding Papers (2016)

Awards of JSE Outstanding Papers (2016)

The complete mitochondrial genome of Gadus chalcogramma and phylogenetic analysis

Here, we report the complete mitochondrial genome sequence of Alaska Pollock, Gadus chalcogramma. The genome sequence was obtained via long PCR reactions using universal primer sets for the fish mitochondrial genome. The total length of mitochondrial genome was 16,571 bp, consisting of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and one control region (D-loop). Except for ND6 and eight tRNA genes, all of the other mitochondrial genes were encoded on the heavy strand. The NJ tree of the combined 13 protein-coding gene sequences of Gadus chalcogramma possesses a relatively closer relationship with the Okhotsk Sea Pollock (Gadus chalcogrammus). Our complete mitochondrial genome will be valuable resources for phylogeny, genetics, and conservation of the Gadus chalcogramma in Korea.