Partial 16S Ribosomal RNA Gene Research Articles

Streptomyces sp. ADR1, Strain Producing β- and γ-Rubromycin Antibiotics, Isolated from Algerian Sahara Desert

A Gram-positive strain, ADR1, was isolated from soil collected from the Algerian Sahara Desert. The ethyl acetate extract of the fermentation broth showed cytotoxic activity against the PANC-1 cell line (37.1 ± 1.3% viability when applied at a concentration of 100 µg/mL). Fractionation and NMR analysis of two peaks absorbing at 490 nm revealed that they represented β- and γ-rubromycin, anticancer antibiotic compounds. The ADR1 strain contained LL-diaminopimelic acid in the whole-cell hydrolysate, and the partial 16S ribosomal RNA gene sequence (1392 bp, Accession No. KF947515) showed 99% sequence similarity to Streptomyces species. Therefore, the name Streptomyces sp. ADR1 was proposed and deposited in the Wellness Industries Culture Collection (WICC) of the Institute of Bioproduct Development, UTM, Malaysia, under the number (WICC- B86). In a 16 L stirred-tank bioreactor, the stain was adapted to submerged culture conditions and produced rubromycins at a relatively high concentration, with maximums of 24.58 mg/L and 356 mg/L for β- and γ-rubromycins, respectively.

Residual Bacteriome after Chemomechanical Preparation of Root Canals in Primary and Secondary Infections

IntroductionSecondary infections may be linked to the presence of residual microorganisms within dental root canals. The purpose of this study was to investigate the bacterial composition of primary and secondary root canal infections before and after chemomechanical treatment. MethodsSamples were collected before chemomechanical preparation (S1) and before obturation (S2) from 19 subjects (10 primary and 9 secondary infections). DNA was extracted, and the V3/V4 region of the 16S ribosomal RNA gene was amplified using the 347 F/803R primers and paired-end sequenced using the MiSeq (Illumina, San Diego, CA) instrument. ResultsSequencing analysis yielded partial 16S ribosomal RNA gene sequences that were taxonomically classified into 10 phyla and 143 genera. The most prevalent phyla in the S1 and S2 samples were Firmicutes and Bacteroides; however, when comparing between sample groups, Proteobacteria seem to have been enriched in secondary infections. The dominant genera in the primary S1 samples were Bacillus, Streptococcus, and Prevotella, whereas Bacillus, Streptococcus, and Selenomonas dominated the secondary infection S1 samples. Bacillus and Marinilactibacillus were the most dominant genera in the primary and secondary S2 samples. The mean number of operational taxonomic units per sample was 32,656 (±12,124 SD) and 37,113 (±16,994 SD) in the S1 and S2 samples, respectively. Alpha and beta diversities presented the same pattern within samples from both groups. ConclusionsGreat interindividual variations in the bacterial composition of the root canal biofilms were observed. There was no difference in the bacterial composition before and after treatment, although some genera survived and seem to be part of a residual microbiome. Our findings revealed a high diversity of the bacterial communities present in root canal infections after chemomechanical treatment, although the majority of the taxa detected were in low abundance.

The bacterial communities of black soldier fly larvae (Hermetia illucens) during consecutive, industrial rearing cycles

In recent years, the (gut) microbiota of black soldier fly larvae (BSFL, Hermetia illucens) has been the focus of several studies that try to elucidate its composition, its interaction with rearing parameters (like the substrate used) and its impact on rearing performance. While these studies have improved our understanding, conclusions from these studies are often based on only one or a few (mostly laboratory) rearing cycles. This leaves the questions whether such results are impacted by variability in between rearing cycles and whether sampling the microbiota, under the same rearing parameters, would yield repeatable results. To obtain insight in the overall variability in the microbiota, the bacterial community compositions of BSFL as well as the feeding substrates were investigated at two different time points during six consecutive rearing cycles in three BSFL-producing companies. This was done by sequencing the partial 16S ribosomal RNA gene amplicons. Based on the results, two main observations were done: (1) there was a clear resemblance over the different cycles at each time point for each individual company, and (2) a clear shift in the community for larvae as well as substrates occurred between the two time points within one production cycle. Additionally, when comparing production sites, an Enterococcus sp. was found at each time point for each company. Other species, such as Corynebacterium sp. and Providencia sp., were commonly found in all companies as well. To conclude, our study reveals that there is a low inter-cycle variability in the larval bacterial community over consecutive rearing cycles within a single company, when they operate using a standardised rearing protocol.

MinION, a portable long-read sequencer, enables rapid vaginal microbiota analysis in a clinical setting

BackgroundIt has been suggested that the local microbiota in the reproductive organs is relevant to women's health and may also affect pregnancy outcomes. Analysis of partial 16S ribosomal RNA (rRNA) gene sequences generated by short-read sequencers has been used to identify vaginal and endometrial microbiota, but it requires a long time to obtain the results, making it unsuitable for rapid bacterial identification from a small specimen amount in a clinical context.MethodsWe developed a simple workflow using the nanopore sequencer MinION that allows high-resolution and rapid differentiation of vaginal microbiota. Vaginal samples collected from 18 participants were subjected to DNA extraction and full-length 16S rRNA gene sequencing with MinION.ResultsThe principal coordinate analysis showed no differences in the bacterial compositions regardless of the sample collection method. The analysis of vaginal microbiota could be completed with a total analysis time of approximately four hours, allowing same-day results. Taxonomic profiling by MinION sequencing revealed relatively low diversity of the vaginal bacterial community, identifying the prevailing Lactobacillus species and several causative agents of bacterial vaginosis.ConclusionsFull-length 16S rRNA gene sequencing analysis with MinION provides a rapid means for identifying vaginal bacteria with higher resolution. Species-level profiling of human vaginal microbiota by MinION sequencing can allow the analysis of associations with conditions such as genital infections, endometritis, and threatened miscarriage.

Species-specific PCR-based marker for rapid detection of Aspidiotus rigidus Reyne (Hemiptera: Diaspididae)

• A marker was designed from InDels of Aspidiotus rigidus 28 s rDNA sequences. • Species-specific delineation of A. rigidus works in singleplex and multiplex PCR. • A. rigidus multiplex-based detection still works with parasitized samples. The Philippine coconut production has been greatly affected by the recent devastating infestation of Aspidiotus spp. However, identification of the outbreak species, Aspidotus rigidus , has been a challenge using morphological approaches. Molecular identification via PCR sequencing of insect barcoding genes has been implemented, but the overall process is time-consuming and costly. Thus, we developed and optimized a species-specific PCR-based molecular marker for rapid, efficient and cost-effective molecular identification of A. rigidus . The molecular marker was designed based on the sequences of the partial 28S ribosomal RNA gene from species of Aspidiotus that feed on coconut in the Philippines, A. rigidus, A. destructor and A. excisus . Multiple alignment of nucleotide sequences revealed a conserved 16-bp insertion-deletion (InDel) site common to all A. rigidus specimens identified from which the A. rigidus- specific oligonucleotide (RIG1) primer targeting an approximately 570 bp fragment size was designed. Results showed that the species-specific DNA marker technology consistently delineated laboratory-reared and field-collected A. rigidus samples from A. destructor and A. excisus . The protocol offers a rapid and reliable method for the early detection of A. rigidus infestation in high-risk areas planted with coconut in the country.

Molecular characterization and phylogenetic analysis of Escherichia coli isolated from milk of cattle affected by mastitis

This evolution-based study aimed to reliably identify the epidemiological prevalence of Escherichia coli that wasrecovered from affected milk of cattle by mastitis, study the evolution of this bacterium, and describe some isolates using polymerase chain reaction (PCR) technique and DNA sequencing. Here, we collected 50 cattle milk samples and submitted them to conventional bacterial isolation and identification using enrichment culture method and biochemical tests. Then, we confirmed the results by PCR technique based on 16S ribosomal RNA gene. The results showed that E. coli was isolated from cattle at (36%), and this was confirmed by PCR that showed highly specific detection of E. coli isolates at (100%). DNA sequencing of partial 16S ribosomal RNA gene showed (99%) homological identity with NCBI-Blast E. coli isolates and the phylogenetic analysis showed genetic similarity (0.5 genetic changes). In conclusion, this was the first study in Iraq to report genetic relationship between E. coli isolated from milk of mastitis-infected cattle. Therefore, it is essential to define the role of animals as an important source in the distribution of some pathogens that are related to public health.

A NEW SPECIES OF MYXOBOLUS (CNIDARIA: MYXOSPOREA: MYXOBOLIDAE) FROM THE BLUE SUCKER, CYCLEPTUS ELONGATUS (LESUEUR) (CYPRINIFORMES: CATOSTOMIDAE: CYCLEPTINAE), FROM ARKANSAS.

During 9-10 February 2018 and 21-22 February 2020, 7 adult Blue Suckers, Cycleptus elongatus, were collected by hoop nets from the Red River, Little River County (n = 3), and the Black River, Lawrence County (n = 4), Arkansas, and their gills, gallbladders, fins, integument, other major organs, and musculature were examined for myxozoans. All 7 (100%) were infected with an unknown species of gill-infecting Myxobolus sp. Twenty formalin-fixed plasmodia (cysts) of Myxobolus cloutmani n. sp. were elliptoidal, 407 μm long × 270 μm wide. Formalin-fixed myxospores were orbicular to broadly elliptoidal, 8.7 μm long × 7.8 μm wide. Two polar capsules were pyriform and subequal in size, extending over halfway in the myxospore. The larger polar capsule was 5.5 μm long × 3.1 μm wide, while the shorter was 5.1 × 2.9 μm. A coiled polar filament possessed 5 or 6 coils. The myxospore was 3.7 μm thick in sutural view, with a distinct sutural ridge. Qualitative and quantitative morphological data were from formalin-fixed as well as ethanol-preserved spores, while molecular data consisted of a 2,010 base pair sequence of the partial 18S ribosomal RNA gene and a 2,502 base pair sequence of the partial 28S ribosomal RNA gene. Phylogenetic analysis grouped M. cloutmani n. sp. with the other catostomid-infecting myxobolids. This is the first myxozoan reported from C. elongatus.

Morphological and molecular characterization of Paratylenchus beltsvillensis n. sp. (Tylenchida: Paratylenchidae) from the rhizosphere of pine tree (Pinus virginiana Mill) in Maryland, USA.

The pin nematode, Paratylechus beltsvillensis n. sp. collected from rhizosphere soil of a Virginia pine tree (Pinus virginiana Mill) growing in Little Paint Branch Park, Beltsville, Prince George’s County, Maryland, USA, is described and illustrated along with light and scanning electron photomicrographs. Females, males, and juveniles of this new species were recovered from soil samples using the sugar centrifugal flotation and Baermann funnel extraction methods. Morphologically, females are short, body length ranging from 245 to 267 μm, stylet from 70 to 75 μm long with anchor shaped knobs, vulva located at 70–73% and small vulval flap, spermatheca large, and ovoid filled with sperms. Lateral field with three incisures, of which the outer two are prominent. Tail slender, having a rounded tail terminus. Males without stylet and have a degenerated pharynx, spicules = 17–20 µm and gubernaculum = 5.0–5.5 µm. Both morphological observations and molecular analysis of ITS and partial 28S ribosomal RNA gene sequences indicated that the specimens collected from the soil at Beltsville Park from rhizosphere soil samples from Virginia pine represents a new pin nematode species.

Bacteriological assessment of tropical retail fresh-cut, ready-to-eat fruits in south-western Nigeria

Globally, more than 20 kinds of fruits are vended as minimally processed or fresh-cut fruits (FCFs). In Nigeria, pineapple (Ananas comosus) and watermelon (Citrullus lanatus) are among the commonest FCFs retailed and consumed regularly. However, the consumption of FCFs vended in open markets may constitute health risks owing to microbial contamination. This study assessed the bacteriological safety of FCFs in urban markets across six south-western Nigerian states. One hundred and twenty pineapple and watermelon products, samples of fruit wash water and vendors’ hand swabs were randomly sampled in markets and analysed for bacterial contamination by standard bacterial culturing methods, followed by analyses of partial 16S ribosomal RNA (rRNA) gene sequences. Bacterial isolates were further screened for haemolysin and amylase production, as well as subjected to antibiotic sensitivity testing against eight commonly administered antibiotics. Citrobacter, Enterobacter, Escherichia, Klebsiella, Pantoea and Shigella were the enteric bacterial genera identified in the fruits. Within these genera, 11 species were detected including two potential pathogens, Enterobacter hormaechei and Enterobacter sichuanensis, that are reported for the first time in vended FCFs in Nigeria. Klebsiella pneumoniae was detected across all sampling points and sample types, suggesting its prevalence in the FCFs process chain. About 10% of 102 isolates obtained had haemolytic potentials against erythrocytes, while 9.8% produced amylase. About 76% of the isolates were multidrug-resistant strains. Co-resistance to ampicillin and gentamicin was the most prevalent resistance pattern observed. About 7% of all isolates including those of Escherichia coli, E. hormaechei, E. sichuanensis and Shigella flexneri were resistant to all the tested antibiotics. Hand swabs from fruit vendors and fruit wash water revealed phylotypes similar to those in the FCFs, suggesting their involvement as potential sources of contamination of the fruits. Appropriate food safety measures for handlers and consumers of FCFs are thus recommended.

Characterization of Pyrenophora Species Causing Brown Leaf Spot on Italian Ryegrass (Lolium multiflorum) in Southwestern China.

Drechslera leaf spot (DLS) caused by Pyrenophora (Drechslera) species is one of the most serious diseases affecting Italian ryegrass (Lolium multiflorum) in China. Between 2015 and 2018, this disease was observed in three Italian ryegrass fields in the province of Sichuan, China. Average leaf disease incidence was approximately 1 to 12% but could range up to 100%. Symptoms appeared as brown or tan spots surrounded by a yellow halo, or brown to dark brown net blotch; subsequently, spots increased in number and size, and they later covered a large area of leaf, eventually causing leaf death. In this study, 86 strains of Pyrenophora fungi were isolated from leaf lesions of Italian ryegrass. Coupled with phylogenetic analysis of the internal transcribed spacer region, partial 28S ribosomal RNA gene, and glyceraldehyde-3-phosphate dehydrogenase gene, morphological characteristics showed that Pyrenophora dictyoides and P. nobleae are associated with Italian ryegrass in southwest China. Pathogenicity tests confirmed that both species can infect Italian ryegrass, causing leaf spot, whereas the virulence of the two species differed; P. nobleae showed lower pathogenicity to Italian ryegrass. This is the first time that these two Pyrenophora species were formally reported on Italian ryegrass based on both morphological and molecular characters. Overall, this study improves knowledge of the Pyrenophora species associated with Italian ryegrass and provides a foundation for control of this disease in the future.

Rapid Detection of Phosphate-Solubilizing Bacteria from Agricultural Areas in Erzurum.

In this study, the newly designed pqq gene-specific primer sets were used for determination of phosphate-solubilizing capabilities of bacterial isolates from the agricultural regions of Erzurum. The specificity of newly designed primer sets (PqqA2F/PqqA2R, Pqq5F/Pqq5R, PqqF2/PqqF2R) were tested against ten isolates, whose phosphate-solubilizing activities were initially proved by the conventional methods. Non-phosphate-solubilizing bacteria were also chosen as negative control. According to the results, five of ten phosphate-solubilizing bacteria with PqqA2F/PqqA2R, two of ten phosphate-solubilizing bacteria with Pqq5F/Pqq5R primer set, and one of ten phosphate solubilizing with PqqF2F/PqqF2R bacteria were successfully amplificated in the PCR assay and none of the non-phosphate-solubilizing bacteria was amplificated. Then, the molecular characterization of the active phosphate-solubilizing strains was done based on the partial 16S ribosomal RNA gene region sequence analysis method. Two isolates of Enterobacter sp., 1 Rhizobium sp., 1 Enterococcus sp., 1 Bacillus cereus, 1 Bacillus atrophaeus, 1 Bacillus aryabhattai, 1 Acinetobacter sp., 1 Pseudomonas japonica, and 1 Enterobacter cloacae were identified as active phosphate-solubilizing strains. Consequently, the results showed that this specific primer sets could be used as an economic, rapid, and useful tool for the detection of phosphate-solubilizing strains in the agricultural researches.

New insight into the species diversity and life cycles of rust fungi (Pucciniales) affecting bioenergy switchgrass (Panicum virgatum) in the Eastern and Central United States

Research was undertaken to clarify the taxonomic identity of leaf rust (Pucciniales) fungi on bioenergy switchgrass in the Eastern and Central U.S. We integrated internal transcribed spacer 2 (ITS2) and partial 28S ribosomal RNA gene sequence data from collections taken from cultivated switchgrass and herbarium specimens, including purported aecial and telial states of Puccinia graminicola and Puccinia pammelii. Maximum likelihood and Bayesian analyses revealed four monophyletic clades: Puccinia emaculata sensu stricto (s.s.), P. pammelii, P. graminicola, and Puccinia novopanici. Results also indicated that P. emaculata s.s. was not affecting cultivated, bioenergy switchgrass. Aecidium pammelii and P. pammelii were distinct phylogenetically from P. emaculata s.s. and grouped within a well-supported clade, demonstrating aecial-telial host alternation for P. pammelii between Euphorbia corollata and switchgrass. Aecidium stillingiae on queen’s delight (Stillingia sylvatica)—a purported aecial state host for P. graminicola—shared identical sequences with the recently described species Puccinia pascua. The latter fungus, however, was recovered within a subclade of P. graminicola. Hence, queen’s delight likely is not an aecial host to P. graminicola s.s. Additional molecular studies are warranted to determine species boundaries within the P. graminicola complex. The majority of contemporary collections from cultivated switchgrass were recognized as P. novopanici. Collectively, bioenergy switchgrass is host to at least three phylogenetically distinct species, presenting a significant challenge to the future selection and breeding of switchgrass with improved rust resistance.

Subcutaneous merocercoids of Clistobothrium sp. in two Cape fur seals (Arctocephalus pusillus pusillus).

Fur seals represent intermediate hosts of the cestode Clistobothrium. Large sharks are definitive hosts for these parasites. Two female, 25– and 27-year-old fur seals, caught in the 1980s at the South African coast, were examined pathomorphologically. Both animals showed multifocal, up to 1 cm in diameter large cavities of the thoracic and abdominal subcutaneous adipose tissue containing intraluminal metacestodes of tapeworms, which were surrounded by a locally extensive, pyogranulomatous panniculitis. The metacestodes (merocercoids) of one fur seal were isolated from the subcutaneous adipose tissue and characterized morphologically and for the first time from this host by molecular techniques. The morphometric data corresponded with ‘delphini'-morphotype merocercoids, but the sequence of the partial 28S ribosomal RNA gene identified them as conspecific with merocercoids of the morphotype ‘grimaldii’. These merocercoid types are morphologically Type XV metacestodes of marine tapeworms and represent different species of Clistobothrium. Sequence data were generated for 18S, ITS1, 5.8S, ITS2, partial 28S ribosomal DNA and partial mitochondrial cox1 gene and phylogenetic analysis of 18S rRNA and partial 28S rRNA genes identified the fur seal merocercoids as Clistobothrium species. However, it cannot yet be assigned to species level because of limited molecular data from adult stages. Most likely, both fur seals were infected as juveniles in their original habitat, the coastal regions of South Africa. The metacestode infection is probably an incidental finding, however, there is a chronic inflammatory reaction next to the subcutaneous merocercoids. It is noteworthy, that the merocercoids remain in a potentially infective stage even after more than 20 years.

A new species of the calcareous sponge genus Leuclathrina (Calcarea: Calcinea: Clathrinida) from the Maldives.

The diversity and phylogenetic relationships of calcareous sponges are still not completely understood. Recent integrative approaches combined analyses of DNA and morphological observations. Such studies resulted in severe taxonomic revisions within the subclass Calcinea and provided the foundation for a phylogenetically meaningful classification. However, several genera are missing from DNA phylogenies and their relationship to other Calcinea remain uncertain. One of these genera is Leuclathrina (family Leucaltidae). We here describe a new species from the Maldives, Leuclathrina translucida sp. nov., which is only the second species of the genus. Like the type species Leuclathrina asconoides, the new species has a leuconoid aquiferous system and lacks a specialized choanoskeleton. Phylogenetic analyses of the partial 28S ribosomal RNA gene revealed that L. translucida sp. nov. is most closely related to a clade containing the exclusively asconoid genera Ascandra, Levinella and Soleneiscus, and to a clade of the likewise asconoid genus Ernstia. No close relationship exists to other members of the polyphyletic family Leucaltidae, or to any other leuconoid Calcinea. Our results suggest that the leuconoid aquiferous system of Leuclathrina evolved independently from that of other calcineans and that the family assignment of the genus has to be reconsidered. Because the latter requires a more comprehensive family level revision among many genera of Calcinea, we propose to formally retain the genus in Leucaltidae for the time being.

Aboveground Whitefly Infestation-Mediated Reshaping of the Root Microbiota.

Plants respond to various types of herbivore and pathogen attack using well-developed defensive machinery designed for self-protection. Infestation from phloem-sucking insects such as whitefly and aphid on plant leaves was previously shown to influence both the saprophytic and pathogenic bacterial community in the plant rhizosphere. However, the modulation of the root microbial community by plants following insect infestation has been largely unexplored. Only limited studies of culture-dependent bacterial diversity caused by whitefly and aphid have been conducted. In this study, to obtain a complete picture of the belowground microbiome community, we performed high-speed and high-throughput next-generation sequencing. We sampled the rhizosphere soils of pepper seedlings at 0, 1, and 2 weeks after whitefly infestation versus the water control. We amplified a partial 16S ribosomal RNA gene (V1–V3 region) by polymerase chain reaction with specific primers. Our analysis revealed that whitefly infestation reshaped the overall microbiota structure compared to that of the control rhizosphere, even after 1 week of infestation. Examination of the relative abundance distributions of microbes demonstrated that whitefly infestation shifted the proteobacterial groups at week 2. Intriguingly, the population of Pseudomonadales of the class Gammaproteobacteria significantly increased after 2 weeks of whitefly infestation, and the fluorescent Pseudomonas spp. recruited to the rhizosphere were confirmed to exhibit insect-killing capacity. Additionally, three taxa, including Caulobacteraceae, Enterobacteriaceae, and Flavobacteriaceae, and three genera, including Achromobacter, Janthinobacterium, and Stenotrophomonas, were the most abundant bacterial groups in the whitefly infested plant rhizosphere. Our results indicate that whitefly infestation leads to the recruitment of specific groups of rhizosphere bacteria by the plant, which confer beneficial traits to the host plant. This study provides a new framework for investigating how aboveground insect feeding modulates the belowground microbiome.

Physiological responses of carbon-sequestering microalgae to elevated carbon regimes

In order to identify a high carbon-sequestering microalgal strain, the physiological effect of different concentrations of carbon sources on microalgae growth was investigated. Five indigenous strains (I-1, I-2, I-3, I-4 and I-5) and a reference strain (I-0: Coccolithus pelagicus 913/3) were subjected to CO2 concentrations of 0.03–15% and NaHCO3 of 0.05–2 g CO2 l–1. The logistic model was applied for data fitting, as well as for estimation of the maximum growth rate (μmax) and the biomass carrying capacity (Bmax). Amongst the five indigenous strains, I-3 was similar to the reference strain with regards to biomass production values. The Bmax of I-3 significantly increased from 214 to 828 mg l–1 when CO2 concentration was increased from 0.03 to 15% (r = 0.955, P = 0.012). Additionally, the Bmax of I-3 increased with increasing NaHCO3 (r = 0.885, P = 0.046) and was recorded at 153 mg l–1 (at 0.05 g CO2 l–1) and 774 mg l–1 at (2 g CO2 l–1). Relative electron transport rate (rETR) and maximum quantum yield (Fv/Fm) were also applied to assess the impact of elevated carbon sources on the microalgal cells at the physiological level. Isolate I-3 displayed the highest rETR confirming its tolerance to higher quantities of carbon. Additionally, the decline in Fv/Fm with increasing carbon was similar for strains I-3 and the reference strain. Based on partial 28s ribosomal RNA gene sequencing, strain I-3 was homologous to the ribosomal genes of Chlorella sp.

First Report of Neoscytalidium dimidiatum Causing Stem and Fruit Canker of Hylocereus undatus in Florida

HomePlant DiseaseVol. 100, No. 7First Report of Neoscytalidium dimidiatum Causing Stem and Fruit Canker of Hylocereus undatus in Florida PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Neoscytalidium dimidiatum Causing Stem and Fruit Canker of Hylocereus undatus in FloridaG. Sanahuja, P. Lopez, and A. J. PalmateerG. SanahujaSearch for more papers by this author, P. LopezSearch for more papers by this author, and A. J. PalmateerSearch for more papers by this authorAffiliationsAuthors and Affiliations G. Sanahuja P. Lopez A. J. Palmateer , Tropical Research and Education Center, University of Florida, Homestead, 33031. Published Online:31 Mar 2016https://doi.org/10.1094/PDIS-11-15-1319-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Pitaya, commonly known as dragon fruit, is a cactus widely grown throughout south Florida for its edible fruit. Two species of pitaya are produced in south Florida: Hylocereus undatus has fruit with white flesh and scarlet colored skin and H. polyrhizus has red flesh with scarlet colored skin. In July 2015, a number of fruit samples of Hylocereus undatus submitted to the Florida Extension Plant Diagnostic Clinic in Homestead, FL, were discovered to have small cankers that appeared as sunken orangish brown spots. Dark gray to black aerial mycelium grew after surface-disinfected lesions were plated on acidified potato dextrose agar (APDA) and incubated at 25°C for 7 days. Colonies produced abundant conidia in arthric chains that were dark brown, cylindrical to round with zero to one-septa and averaged 9.29 ± 1.56 μm × 5.93 ± 0.66 μm (Crous et al. 2006). Genomic DNA was extracted from mycelium and partial 18S ribosomal RNA gene, complete internal transcribed spacer (ITS) 1, 5.8S ribosomal RNA gene, ITS 2, and partial 28S ribosomal RNA gene was amplified with the primers ITS1 and ITS4 and the PCR product was sequenced. The resulting sequence was deposited in GenBank (Accession No. KT803701). Based on morphological characteristics and the ITS sequence region, the pathogen was identified as Neoscytalidium dimidiatum. A BLAST search exhibited 97% nucleotide identity with an isolate of Neoscytalidium dimidiatum that caused pitaya canker disease in China (Accession No. JX524168). To further support identification, phylogenetic analysis by the maximum likelihood method (Tamura-Nei model) was performed (MEGA 6.06) and our isolate clustered with other N. dimidiatum isolates with 74% support (1000 replicates). Pathogenicity tests were conducted inoculating healthy stem and fruit of both species (H. undatus and H. polyrhizus) with 2 mm agar plugs from a 5-day old APDA culture. Two noninoculated controls were included; one with 2-mm agar plugs and one without. Five fruit and five stems were used per treatment and the experiment was repeated. Inoculated fruit and stems were placed in a greenhouse where temperature ranged from 25 to 32°C. Seven days post inoculation, both stem and fruit tissue developed small circular sunken light-brown spots. Neoscytalidium dimidiatum was isolated from all symptomatic tissue confirming Koch’s postulates. No symptoms developed on the noninoculated fruit and stem controls. This disease has been reported in Taiwan, Malaysia, and China (Chuang et al. 2012; Mohd et al. 2013; Lan and He 2012), but this is the first report of N. dimidiatum causing stem and fruit canker on Hylocereus undatus in Florida. It is important to note that Hylocereus polyrhizus is also susceptible to N. dimidiatum. Disease incidence in several local fields has been as high as 70% and appears to be more severe on fruit than stems. Very few fungicides are currently labeled for use on pitaya in Florida, so this disease has been a serious issue for pitaya growers in south Florida.

Isolation of Propionibacterium acnes among the microbiota of primary endodontic infections with and without intraoral communication.

ObjectivesThe presence of opportunistic pathogens such as Propionibacterium acnes (P. acnes) may contribute to the endodontic pathology. The presence of P. acnes may be influenced by different endodontic conditions.The aims of the study were firstly, to identify P. acnes within the whole cultivable microbiota of primary endodontic infections, to investigate which P. acnes phylotypes predominate in such infections and secondly to determine if the presence of an “open” communication (e.g. a sinus) can be associated with the isolation of P. acnes from the root canal.Material and methodsThe predominant cultivable microbiota of 15 primary endodontic lesions (7 without communication with the oral environment and 8 with an open communication) were identified using partial 16S ribosomal RNA (rRNA) gene sequence analysis. The identification of the organism was determined by interrogating the Human Oral Microbiome Database. The P. acnes isolates were typed on the basis of the recA gene sequence comparison. A neighbor-joining tree was constructed using MEGA 4.1 with the inclusion of known recA sequences.ResultsThere was no difference in the number of species identified from lesions without communication (5.86 ± 3.7) and those with communication (5.37 ± 3.6) (P > 0.05). PCR-based 16S rRNA gene sequencing revealed P. acnes as the most prevalent isolate recovered from lesions with communication. recA gene sequencing revealed two phylogenetic lineages present in lesion with communication, with mainly type I (further split into type IA and type IB) and type II.ConclusionsThe presence of P. acnes as opportunistic pathogens has been confirmed and may sustain the traits observed in specific clinical presentations.Clinical relevanceClinical management of open lesions may require further disinfection to eliminate opportunistic bacteria.

Discovering Hidden Diversity of Characins (Teleostei: Characiformes) in Ecuador's Yasuní National Park.

BackgroundManagement and conservation of biodiversity requires adequate species inventories. The Yasuní National Park is one of the most diverse regions on Earth and recent studies of terrestrial vertebrates, based on genetic evidence, have shown high levels of cryptic and undescribed diversity. Few genetic studies have been carried out in freshwater fishes from western Amazonia. Thus, in contrast with terrestrial vertebrates, their content of cryptic diversity remains unknown. In this study, we carried out genetic and morphological analyses on characin fishes at Yasuní National Park, in eastern Ecuador. Our goal was to identify cryptic diversity among one of the most speciose fish families in the Amazon region. This is the first time that genetic evidence has been used to assess the species content of the Napo Basin, one of the richest regions in vertebrate diversity.ResultsPhylogenetic analyses of partial mitochondrial 16S ribosomal RNA gene (∼600 pb) DNA sequences from 232 specimens of the family Characidae and its closest groups revealed eight candidate new species among 33 species sampled, representing a 24% increase in species number. Analyses of external morphology allowed us to confirm the species status of six of the candidate species.ConclusionsOur results show high levels of cryptic diversity in Amazonian characins. If this group is representative of other Amazonian fish, our results would imply that the species richness of the Amazonian ichthyofauna is highly underestimated. Molecular methods are a necessary tool to obtain more realistic inventories of Neotropical freshwater fishes.

Synergistic hemolysis between β-lysin-producing Staphylococcus species and Rothia nasimurium in primary cultures of clinical specimens obtained from dogs.

Nonhemolytic isolates of catalase-positive, Gram-positive cocci were observed that produced strong synergistic hemolysis with colonies of Staphylococcus pseudintermedius and Staphylococcus schleiferi subsp. coagulans in primary cultures of clinical specimens from dogs. The isolates appeared similar to nonhemolytic staphylococci but were not identified with conventional biochemical tests or commercial biochemical test kits. Partial 16S ribosomal RNA gene sequences from each of 9 tested isolates were nearly identical to that of Rothia nasimurium. The isolates produced positive Christie-Atkins-Munch-Peterson test reactions with Staphylococcus aureus (ATCC strain 25923) and Staphylococcus intermedius Hajek (ATCC strain 49052). The latter was identified in the current study as S. pseudintermedius. Rothia nasimurium appears to be a previously unreported commensal organism in dogs. Its potential significance as an opportunistic contributor to polymicrobial infections in dogs is not known.