rRNA ITS Region Research Articles

An integrative approach to the study of Xiphinema brevicolle Lordello and Da Costa 1961, supports its limited distribution worldwide (Nematoda: Longidoridae)

Xiphinema americanum group has a cosmopolitan distribution, with several species having particular importance as virus-vectors of four economically important nepoviruses naturally occurring in the USA with IAI quarantine status for Europe. The morphological diagnosis of the species is problematic due to the limited diagnostic characters, overlapping morphometrics and incomplete taxonomic descriptions. An ongoing issue within the X. americanum group is the determination of species belonging to the X. brevicolle subgroup. By using a phylogenetic framework, molecular and morphometric data of the topotype population we demonstrate that certain of the published DNA sequences have been misidentified and belong to closely related species such as X. taylori, X. diffusum or unknown/unidentified species. Studying again the morphology and morphometrics of populations identified as X. brevicolle from the Czech Republic the identity, phylogenetic position and genetic variability of these populations have been clarified. These belong to a different species that is described herein as X. parataylori n. sp. Multi-gene phylogenies were reconstructed using nuclear ribosomal (18S rRNA gene, ITS1 and ITS2 regions, D2-D3 expansion segments of 28S rRNA gene) and mitochondrial DNA (partial cox1 and nad4) and the position of X. brevicolle (topotype population) to the most closely related species is shown. Morphology and species specific sequences were linked and the worldwide distribution discussed. The validity of X. brevicolle and the long-standing controversies about the taxonomic status of X. diffusum and X. taylori have been clarified. Xiphinema taylori and X. diffusum may represent complexes of cryptic species, however more comprehensive studies involving additional populations and materials from the type localities are needed to support this hypothesis.

Cheese brines from Danish dairies reveal a complex microbiota comprising several halotolerant bacteria and yeasts

The Danish Danbo cheese is a surface ripened semi-hard cheese, which before ripening is submerged in brine for up to 24 h. The brining is required in order to obtain the structural and organoleptic properties of the cheeses. Likewise, the content of NaCl in the cheese will influence especially the surface microbiota being of significant importance for flavour development and prevention of microbial spoilage. Even though the microbiota on cheese surfaces have been studied extensively, limited knowledge is available on the occurrence of microorganisms in cheese brine. The aim of the present study was to investigate by both culture-dependent and -independent techniques the brine microbiota in four Danish dairies producing Danbo cheese. The pH of the brines varied from 5.1 to 5.6 with a dry matter content from 20 to 27% (w/w). The content of lactate varied from 4.1 to 10.8 g/L and free amino acids from 65 to 224 mg/L. Bacteria were isolated on five different media with NaCl contents of 0.85–23.0% (w/v) NaCl. The highest count of 6.3 log CFU/mL was obtained on TSA added 4% (w/v) NaCl. For yeasts, the highest count was 3.7 log CFU/mL on MYGP added 8% (w/v) NaCl. A total of 31 bacterial and eight eukaryotic species were isolated including several halotolerant and/or halophilic species. Among bacteria, counts of ≥6.0 log CFU/mL were obtained for Tetragenococcus muriaticus and Psychrobacter celer, while counts between ≥4.5 and < 6.0 log CFU/mL were obtained for Lactococcus lactis, Staphylococcus equorum, Staphylococcus hominis, Chromohalobacter beijerinckii, Chromohalobacter japonicus and Microbacterium maritypicum. Among yeasts, counts of ≥3.5 log CFU/mL were only obtained for Debaryomyces hansenii. By amplicon-based high-throughput sequencing of 16S rRNA gene and ITS2 regions for bacteria and eukaryotes respectively, brines from the same dairy clustered together indicating the uniqueness of the dairy brine microbiota. To a great extent the results obtained by amplicon sequencing fitted with the culture-dependent technique though each of the two methodologies identified unique genera/species. Dairy brine handling procedures as e.g. microfiltration were found to influence the brine microbiota. The current study proves the occurrence of a specific dairy brine microbiota including several halotolerant and/or halophilic species most likely of sea salt origin. The importance of these species during especially the initial stages of cheese ripening and their influence on cheese quality and safety need to be investigated. Likewise, optimised brine handling procedures and microbial cultures are required to ensure an optimal brine microbiota.

Molecular identification of Trichomonas tenax in the oral environment of domesticated animals in Poland - potential effects of host diversity for human health.

The protozoan Trichomonas tenax is considered to be a human specific flagellate of the oral cavity, found in humans with poor oral hygiene and advanced periodontal disease. Morphological variability and great similarity between species occurring in humans and animals, complicate the specific identification of trichomonads, using microscopic examination and other standard parasitological techniques. The aim of the study was to search for and identify T. tenax in domesticated animals using molecular methods. The obtained data were assessed in terms of potential effects of a spread of the species deriving from the animals in the human environment. 301 animals: 142 dogs, 57 cats and 102 horses, were examined in terms of their mouth status and occurrence of trichomonads. ITS1-5.8S rRNA-ITS2 region was amplified and sequenced. Finally, 7 dogs, 3 cats and 1 horse were diagnosed positive for T. tenax by PCR. In the oral cavity of 9 /11 animals, gingivitis and dental plaque accumulation were diagnosed. 9 /11 sequences of trichomonad isolates showed 100% identity with T. tenax sequence derived from the GenBank. The sequences of 2 isolates differed by substitutions. It was proved that T. tenax, considered so far as a human specific parasite, can also inhabit the oral cavity of dog, cat and horse. To summarize, T. tenax was detected in the mouths of different domesticated animals, indicating that in Poland it can colonize a wider range of hosts than previously known. The owners of 3 dogs showed oral tissue inflammation of different intensity and were also positive for T. tenax; therefore, oral trichomonosis spread from humans to domestic animals and conversely should be taken into consideration.

Two new Ceratobasidium species causing white thread blight on tropical plants in Brazil

White thread blight is one of the most common diseases affecting several tree species that grow in warm and humid regions. The typical symptoms of this disease include blighted leaves hanging by a white thread of fungal hyphae. Leaf samples exhibiting white thread blight symptoms were collected from neem (Azadirachta indica A. Juss) and Brazilian cherry pitanga (Eugenia uniflora L.) plants in Northeastern Brazil, and from Indian green-tea (Camellia sinensis (L.) O. Kuntze), coffee (Coffea arabica L.), and persimmon (Diospyros kaki L.) in agricultural areas neighboring the Atlantic forest in Southeastern Brazil. Fungal isolates were obtained indirectly from leaf fragments or directly by transferring mycelia and sclerotia to culture medium. Bright field and scanning electron microscopy images revealed the association of Rhizoctonia-like hyphae and basidiospores with the infected leaves. In pathogenicity tests, Rhizoctonia-like fungal isolates induced leaf necrosis on their hosts, and the pathogens were re-isolated from inoculated plants. Phylogenetic analyses based on sequences of the ITS rRNA region indicated the occurrence of Ceratobasidium lineages distinct from previously reported Ceratobasidium species. Our study leads to the description of two new species of Ceratobasidium: the fungal isolates from A. indica, C. sinensis, and E. uniflora were classified as Ceratobasidium niltonsouzanum sp. nov., and those obtained from C. arabica and D. kaki as Ceratobasidium chavesanum sp. nov.

Cultivable Microbial Diversity Associated With Cellular Phones.

A substantial majority of global population owns cellular phones independently to demographic factors like age, economic status, and educational attainment. In this study, we investigated the diversity of microorganisms associated with cellular phones of 27 individuals using cultivation-based methods. Cellular phones were sampled using cotton swabs and a total of 554 isolates representing different morphotypes were obtained on four growth media. Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry could generate protein profiles for 527 isolates and species-level identification was obtained for 415 isolates. A dendrogram was constructed based on the protein profiles of the remaining isolates, to group 112 isolates under 39 different proteotypes. The representative strains of each group were selected for 16S rRNA gene and ITS region sequencing based identification. Staphylococcus, Bacillus, Micrococcus, and Pseudomonas were the most frequently encountered bacteria, and Candida, Aspergillus, Aureobasidium, and Cryptococcus were in case of fungi. At species-level the prevalence of Micrococcus luteus, Staphylococcus hominis, Staphylococcus epidermidis, Staphylococcus arlettae, Bacillus subtilis, and Candida parapsilosis was observed, most of these species are commensal microorganisms of human skin. UPGMA dendrogram and PCoA biplot generated based on the microbial communities associated with all cellular phones exhibited build-up of specific communities on cellular phones and the prevalence of objectionable microorganisms in some of the cellular phones can be attributed to the poor hygiene and sanitary practices. The study also revealed the impact of MALDI-TOF MS spectral quality on the identification results. Overall MALDI-TOF appears a powerful tool for routine microbial identification and de-replication of microorganisms. Quality filtering of MALDI-TOF MS spectrum, development of better sample processing methods and enriching the spectral database will improve the role of MALDI-TOF MS in microbial identifications.

Schistosomes and Microfilarial Parasites in Magellanic Penguins.

The Magellanic Penguin ( Spheniscus magellanicus) is native to Argentina, Chile, and the Falkland/Malvinas Islands, and is a regular winter migrant in Uruguayan and Brazilian coastal waters. The species is known to be susceptible to a variety of gastrointestinal nematodes, cestodes, trematodes, and acanthocephalans, as well as renal trematodes and pulmonary nematodes. Schistosomes (Platyhelminthes, Trematoda, Schistosomatidae) and microfilariae (Nematoda, Secernentea, Onchocercidae) were histologically identified in Magellanic Penguins ( Spheniscus magellanicus) that died while under care at rehabilitation centers in southern Brazil. Phylogenetic analysis of the COI gene, ITS-1 region, 5.8S rRNA gene, ITS-2 region, and 28S rRNA gene sequences of the schistosome revealed that it is closely related to, but distinct from, a schistosome reported from the African Penguin ( Spheniscus demersus). The schistosomes from Magellanic and African Penguins were grouped with Gigantobilharzia huronensis, Gigantobilharzia melanoidis, and Dendritobilharzia pulvurenta; however, the lack of a clearly monophyletic origin precludes determining their genus. The incidental discovery of novel parasites during a study that did not specifically aim to investigate the occurrence of helminths underscores the value of histopathological examination as an exploratory diagnostic approach.

Social status shapes the bacterial and fungal gut communities of the honey bee

Despite the fungal abundance in honey and bee bread, little is known about the fungal gut community of the honey bee and its effect on host fitness. Using pyrosequencing of the 16S rRNA gene and ITS2 region amplicons, we analysed the bacterial and fungal gut communities of the honey bee as affected by the host social status. Both communities were significantly affected by the host social status. The bacterial gut community was similar to those characterised in previous studies. The fungal gut communities of most worker bees were highly dominated by Saccharomyces but foraging bees and queens were colonised by diverse fungal species and Zygosaccharomyces, respectively. The high fungal density and positive correlation between Saccharomyces species and Lactobacillus species, known yeast antagonists, were only observed in the nurse bee; this suggested that the conflict between Saccharomyces and Lactobacillus was compromised by the metabolism of the host and/or other gut microbes. PICRUSt analysis revealed significant differences in enriched gene clusters of the bacterial gut communities of the nurse and foraging bees, suggesting that different host social status might induce changes in the gut microbiota, and, that consequently, gut microbial community shifts to adapt to the gut environment.

Metagenomic investigation of vestimentiferan tubeworm endosymbionts from Mid-Cayman Rise reveals new insights into metabolism and diversity

BackgroundThe microbial endosymbionts of two species of vestimentiferan tubeworms (Escarpia sp. and Lamellibrachia sp.2) collected from an area of low-temperature hydrothermal diffuse vent flow at the Mid-Cayman Rise (MCR) in the Caribbean Sea were characterized using microscopy, phylogenetic analyses, and a metagenomic approach.ResultsBacteria, with a typical Gram negative cell envelope contained within membrane-bound vacuoles, were observed within the trophosome of both tubeworm species. Phylogenetic analysis of the 16S rRNA gene and ITS region suggested MCR individuals harbored highly similar endosymbionts that were > 98% identical, with the exception of two symbionts that showed a 60 bp insertion within the ITS region. All sequences from MCR endosymbionts formed a separate well-supported clade that diverged from those of symbionts of seep and vent vestimentiferans from the Pacific, Gulf of Mexico, and Mediterranean Sea. The metagenomes of the symbionts of two specimens of each tubeworm species were sequenced, and two distinct Gammaproteobacteria metagenome-assembled genomes (MAGs) of more than 4 Mbp assembled. An Average Nucleotide Identity (ANI) of 86.5% between these MAGs, together with distinct 16S rRNA gene and ITS sequences, indicate the presence of multiple endosymbiont phylotypes at the MCR, with one MAG shared between one Escarpia and two Lamellibrachia individuals, indicating these endosymbionts are not specific to either host species. Genes for sulfur and hydrogen oxidation, nitrate reduction (assimilatory and dissimilatory), glycolysis and the Krebs cycle, peptide, sugar, and lipid transporters, and both rTCA and CBB carbon fixation cycles were detected in the MAGs, highlighting key and shared functions with symbiont metagenomes of the vestimentiferans Riftia, Tevnia, and Ridgeia from the Pacific. The potential for a second hydrogen oxidation pathway (via a bidirectional hydrogenase), formate dehydrogenase, a catalase, and several additional peptide transporters were found exclusively in the MCR endosymbiont MAGs.ConclusionsThe present study adds new evidence that tubeworm endosymbionts can potentially switch from autotrophic to heterotrophic metabolism, or may be mixotrophic, presumably while free-living, and also suggests their versatile metabolic potential may enable both the host and symbionts to exploit a wide range of environmental conditions. Together, the marked gene content and sequence dissimilarity at the rRNA operon and whole genome level between vent and seep symbionts suggest these newly described endosymbionts from the MCR belong to a novel tubeworm endosymbiont genera, introduced as Candidatus Vondammii.

Phylogenetic analysis and assessment ability of Fusarium isolates for the biodegradation of the pesticide carbofuran

Carbofuran is a pesticide used against insects and nematodes. It is a common environmental pollutant associated with agricultural activities. Carbofuran has harmful effects on target and non-target organisms through the accumulation of acetylcholine at the junction of the nerve cell and the receptor sites system by inhibiting acetylcholine esterase. Since the Kingdom of Saudi Arabia is one of the countries that use the pesticide carbofuran in agricultural applications, it inevitably suffers from this worsening environmental problem. Therefore, removal of this compound from the environment is a necessity to ensure human health. In this study, 20 fungal isolates were isolated from pesticide-contaminated agricultural soils. Three fungal isolates (KKU-F5, KKU-F11, KKU-F17) were selected that have the ability to tolerate and utilise carbofuran. The exact identification of these isolates was determined using ITS1-5.8S rRNA-ITS2 region amplification and sequence determination. Alignment results and the comparison of ITS1-5.8S rRNA-ITS2 region sequences of the isolates to ITS1-5.8S rRNA-ITS2 sequences available in the GenBank database, as well as the phylogenetic analysis, confirmed the accurate identification of the isolates KKU-F5, KKU-F11 and KKU-F17 as Fusarium polyphialidicum, Fusarium oxysporum and Fusarium verticillioides, respectively. The three selected fungi demonstrated a high growth rate on 3 gm/L carbofuran, which is twice the recommended dose of carbofuran, after an incubation period of seven days. The percentage of carbofuran removal determined by HPLC analysis for the isolates KKU-F 5, KKU-F11 and KKU-F17 was 88%, 77% and 90.10% respectively, within this period. RAPD-PCR fingerprinting was performed to determine the differentiation at the molecular level within the three Genetics and Molecular Research 17 (1): gmr16039900 carbofuran-degrading Fusarium isolates (KKU-F5, KKU-F11 and KKU-F17). The RAPD-PCR results exhibited different RAPD banding profiles indicating that the three Fusarium isolates were different from each other and could be clearly distinguished by their amplification patterns.

Occurrence of Toxigenic Fungi and Mycotoxins during Smoked Paprika Production

'La Vera' smoked paprika is a traditional Spanish product regulated under a protected designation of origin. Mycotoxins are possible contaminants in paprika, yet there is little information about mycotoxin production during the processing of smoked paprika. In this study, samples of dried peppers collected from six traditional dryers from four producers were evaluated for physicochemical parameters, mycotoxins, and mycotoxin-producing fungi. The moisture content and water activity of the peppers ranged from 11.0 to 16.3% and 0.513 to 0.611, respectively, with significant differences among the dryers (P ≤ 0.05). Culture methods revealed fungal counts of 2.6 to 5.7 log CFU/g, with significant differences among the dryers (P ≤ 0.05), and real-time PCR revealed aflatoxin-producing fungi (2.00 to 3.42 log CFU/g) in all dryers. However, mycotoxins were not detected in dried pepper samples. Sixty-seven mold species isolated from dried peppers were identified by sequencing of the ITS1-5.8S rRNA-ITS2 region and characterized by mycotoxigenic ability. Four isolates of Penicillium expansum, four isolates of Penicillium thomii, and one isolate of Aspergillus parasiticus were producers of patulin, penicillic acid, and aflatoxins, respectively. Toxigenic fungi were inoculated onto smoked dried peppers and stored at 84, 91, 94, and 97% relative humidity (RH) at 20°C for 30 days. Patulin was not detected under any of these conditions. Penicillic acid was detected in dried samples stored at 91 to 97% RH, although the optimum condition was isolate dependent. Aflatoxins G2, B1, and B2 were detected at 91 to 97% RH, with the highest concentrations at 94% RH. According to our results, hazard analysis critical control point systems should be used to control the drying and storage conditions of dried peppers until the milling step to avoid rehydration, which encourages fungal growth and mycotoxin production.

Erysiphe azerbaijanica and E. linderae: Two new powdery mildew species (Erysiphales) belonging to the Microsphaera lineage of Erysiphe

Two new species, Erysiphe azerbaijanica on Castanea sativa and E. linderae on Lindera praecox, both belonging to the Microsphaera lineage of the genus Erysiphe are described based on morphological and molecular data. Erysiphe azerbaijanica is distinguished from other Erysiphe species occurring on Castanea spp. by its cylindrical conidia with a length/width ratio of 2–3.6, longer conidiophore, and foot-cells. Molecular analyses indicated that this species forms a clade of its own, supporting the morphological observations. Molecular phylogenetic analyses showed that E. blasti s. lat. is divided into two genetically differentiated groups associated with different host species. Based on the sequence differences in the 28S rRNA gene and ITS region, connected with differences in the number and length of appendages, the fungus on L. praecox is described as a new species, E. linderae.

First Report of Common Scab on Potato Caused by Streptomyces europaeiscabiei in Taiwan

HomePlant DiseaseVol. 102, No. 4First Report of Common Scab on Potato Caused by Streptomyces europaeiscabiei in Taiwan PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Common Scab on Potato Caused by Streptomyces europaeiscabiei in TaiwanC. Y. Lin, H. F. Ni, and C. W. HuangC. Y. Lin†Corresponding author: C. Y. Lin; E-mail: E-mail Address: eris2024@dns.caes.gov.twSearch for more papers by this author, H. F. NiSearch for more papers by this author, and C. W. HuangSearch for more papers by this authorAffiliationsAuthors and Affiliations C. Y. Lin † H. F. Ni C. W. Huang , Department of Plant Protection, Chiayi Agricultural Experiment Branch, Agricultural Research Institute, Chiayi 60014, Taiwan. Published Online:12 Feb 2018https://doi.org/10.1094/PDIS-05-17-0667-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Common scab caused by Streptomyces spp. is an economically important disease of potato (Solanum tuberosum L.) worldwide. In Taiwan, only Streptomyces scabies has been reported as the causal species of common scab on potato tubers. In spring of 2015 and 2016, potato tubers of cv. Kennebec with scab symptoms were collected from 14 fields in Taichung, Yunlin County, and Chiayi County. Bacteria were isolated from scab lesions using International Streptomyces Project (ISP) medium 4. Colonies with white to gray spores were transferred to a new ISP medium 4 after 7 days. Pure cultures of isolates were obtained by transferring single colonies three times. Genomic DNA of pure culture bacteria was extracted by an NaOH-based method (Wang et al. 1993) and used as template for PCR with species-specific primers for 16S rRNA gene sequences of S. scabies, S. turgidiscabiei, and S. acidiscabiei (Leiminger et al. 2013). Of the 33 bacterial isolates recovered, 31 isolates had amplified segments of S. scabies and two remained unidentified. Because 16S rRNA gene sequences in S. scabies and S. europaeiscabiei are nearly identical, distinguishing the species requires exploitation of an Hpy99I endonuclease restriction site in the 16S-23S rRNA ITS region of S. scabies (Flores-González et al. 2008). Among the 31 Streptomyces spp. isolates tested, one isolate could not be cleaved with Hpy99I and consequently was identified as S. europaeiscabiei. To further confirm the identity of the isolate, sequences of atpD, recA, and ropB genes (GenBank accession nos. KX981452, KX981453, and KX981454) from the isolate were found to be 99.4, 100, and 99.9% identical, respectively, to corresponding S. europaeiscabiei type strain IBSBF 2023 (JF303745) and S. europaeiscabiei strain 364 (KJ802472 and KJ802473) sequences (Hiltunen et al. 2014). Pathogenicity of the S. europaeiscabiei isolate was confirmed using a radish seedling assay (Flores-González et al. 2008). The radish seedlings grown with the S. europaeiscabiei isolate showed reduced growth and hypertrophy compared with seedlings grown on oat meal agar without bacteria. In addition, potato seedlings (cv. Kennebec) (three pots per treatment) was inoculated with a spore suspension (106 CFU/ml) of the S. europaeiscabiei isolate. Six weeks later, potato tubers were harvested and evaluated for scab symptoms. The experiment was done twice. Potato tubers from the plants inoculated with S. europaeiscabiei isolate exhibited superficial, raised, or pitted scab symptoms, whereas no symptoms were observed on tubers from the noninoculated potato plants. The pathogen was reisolated from the lesions and confirmed identical to the original isolate by PCR and Hpy99I digestion. To our knowledge, this is the first report of S. europaeiscabiei causing potato common scab in Taiwan.

Jaminaea pallidilutea sp. nov. (Microstromatales), a basidiomycetous yeast isolated from plant material of mangrove forests in Iran.

In the course of an ongoing study aiming to catalogue the natural yeast biodiversity of Iran, a number of yeasts were isolated from plant material collected from mangrove forests on the shoreline of Qeshm Island. Two strains were identified as members of order Microstromatales. Standard phenotypic, biochemical, physiological characterization and a phylogenetic analyses of the combined 26S rRNA gene (D1/D2 domains) and ITS region sequences showed the conspecificity of these isolates and suggest their placement in the genus Jaminaea, close to Jaminaea lanaiensis and Jaminaea angkoriensis. Here, we describe this species as Jaminiaea pallidilutea sp. nov. with IBRC-M 30284T=DSM 104392T=CBS 14684T as the type strain. The Mycobank accession number is MB 819618.

Molecular and morphological characterization of the spiral nematode Helicotylenchus oleae Inserra, Vovlas &amp; Golden, 1979 (Nematoda: Hoplolaimidae) in the Mediterranean Basin

The spiral nematode Helicotylenchus oleae is an ectoparasite of olive roots and is distributed in some countries in the Mediterranean Basin. In this study, we provided morphological and molecular characterisation of topotypes from southern Italy, as well as of several other populations from Crete (Greece) and Spain. Correct identification of plant-parasitic nematode species is essential to establish appropriate control strategies and for preventing their spread to other areas. Helicotylenchus oleae is reported for the first time in Greece (Crete). Integrative morphometric and molecular data for H. oleae populations using D2-D3 expansion segments of 28S rDNA, ITS-rDNA, and the cytochrome c oxidase subunit 1 (coxI), were in agreement with the original descriptions of the species, except for some minor differences, which may be a result of intraspecific variability. The phylogenetic relationships of this species with other representatives of Helicotylenchus spp. using D2-D3 expansion segments and the ITS-rRNA region was studied showing a low intraspecific diversity for H. oleae species. For the first time, coxI molecular data is obtained for the genus Helicotylenchus.

Prevalence of Centrocestus formosanus Metacercariae in Ornamental Fish from Chiang Mai, Thailand, with Molecular Approach Using ITS2.

The prevalence of Centrocestus formosanus metacercariae was investigated in ornamental fish purchased from a pet shop in Chiang Mai, Thailand, including Carassius auratus (goldfish), Cyprinus carpio (Koi), Poecilia latipinna (Sailfin Molly), Danio rerio (Zebrafish), and Puntigrus tetrazona (Tiger barb). The parasite species was identified by the morphology of worms as well as by a molecular approach using ITS2. The results showed that 50 (33.3%) of 150 fish examined were infected with the metacercariae. The highest prevalence was found in C. auratus (83.3%), and the highest intensity was noted in C. carpio (70.8 metacercariae/fish). The most important morphological character was the presence of 32–34 circumoral spines on the oral sucker. The phylogenetic studies using the rRNA ITS2 region revealed that all the specimens of C. formosanus in this study were grouped together with C. formosanus in GenBank database. This is the first report on ornamental fish, C. carpio, P. latipinna, D. rerio, and P. tetrazona, taking the role of second intermediate hosts of C. formosanus in Thailand. Prevention and control of metacercarial infection in ornamental fish is urgently needed.

Biogeography and organic matter removal shape long-term effects of timber harvesting on forest soil microbial communities.

The growing demand for renewable, carbon-neutral materials and energy is leading to intensified forest land-use. The long-term ecological challenges associated with maintaining soil fertility in managed forests are not yet known, in part due to the complexity of soil microbial communities and the heterogeneity of forest soils. This study determined the long-term effects of timber harvesting, accompanied by varied organic matter (OM) removal, on bacterial and fungal soil populations in 11- to 17-year-old reforested coniferous plantations at 18 sites across North America. Analysis of highly replicated 16 S rRNA gene and ITS region pyrotag libraries and shotgun metagenomes demonstrated consistent changes in microbial communities in harvested plots that included the expansion of desiccation- and heat-tolerant organisms and decline in diversity of ectomycorrhizal fungi. However, the majority of taxa, including the most abundant and cosmopolitan groups, were unaffected by harvesting. Shifts in microbial populations that corresponded to increased temperature and soil dryness were moderated by OM retention, which also selected for sub-populations of fungal decomposers. Biogeographical differences in the distribution of taxa as well as local edaphic and environmental conditions produced substantial variation in the effects of harvesting. This extensive molecular-based investigation of forest soil advances our understanding of forest disturbance and lays the foundation for monitoring long-term impacts of timber harvesting.

Morphological, ultrastructural, and molecular characterization of intestinal tetratrichomonads isolated from non-human primates in southeastern Brazil.

Non-human primates are our closest relatives and represent an interesting model for comparative parasitological studies. However, research on this topic particularly in relation to intestinal parasites has been fragmentary and limited mainly to animals held in captivity. Thus, our knowledge of host-parasite relationships in this species-rich group of mammals could be considered rudimentary. The current study combined morphological, ultrastructural, and molecular analyses to characterize isolates of intestinal tetratrichomonads recovered from the feces of three species of South American, non-human primates. Fecal samples were collected from 16 animals, representing 12 distinct species. Parabasalid-like organisms were evident in five samples (31%) of feces: two from Alouatta sara, two from Callithrix penicillata, and one from Sapajus apella. The five samples presented morphologies consistent with the description of Tetratrichomonas sp., with four anterior flagella of unequal length, a well-developed undulating membrane, and a long recurrent flagellum. Sequencing of the ITS1-5.8S rRNA-ITS2 region demonstrated that the isolates from A. sara, and C. penicillata were closely related and highly similar to isolates of Tetratrichomonas brumpti, recovered previously from tortoises (Geochelone sp.). The flagellate recovered from S. apella demonstrated a similar morphology to those of the other isolates, however, sequence analysis showed it to be identical to an isolate of Tetratrichomonas sp. recovered from white-lipped peccaries (Tayassu pecari). The findings of this study extend and enhance our knowledge of parasitism of non-human primates by members of the genus Tetratrichomonas and indicate that the host range of these parasites is broader than previously believed.

Identification of the fungus Erysiphe diffusa causing powdery mildew disease on soybeans

Fungus Erysiphe diffusa Cooke &amp; Peck 2000, which causes powdery mildew disease on soybeans (Glycine max (L) Merr) cultivated in Vinh Phuc Province, has already been identified using a combination of molecular biotechnology and an updated standard morphological classification for the confirmation of a pathogenicity test to fulfill Koch's postulate. Six sequences of the ITS rRNA region of this fungus on soybeans have already been registered in GenBank with accession numbers including KM260706 and KM260708 - KM260712.

Hookworm infections among migrant workers in Malaysia: Molecular identification of Necator americanus and Ancylostoma duodenale

Ongoing urbanisation of the working population as well as cross-border migration of workers particularly into large cities has contributed to the development and growth of urban slums. These deprived areas are conducive for the transmission of intestinal pathogens including hookworm. The aim of this study was to determine both the prevalence and species identity of hookworm infections among the migrant worker community in Malaysia. A total of 388 faecal samples were collected from migrant workers between September 2014 and August 2015, representing workers from five employment sectors: construction, manufacturing, agriculture and plantations, food services and domestic services. Faecal samples were examined by microscopy and positive samples were subjected to molecular analysis. A total of 51 samples (13.1%) were positive by microscopy for hookworm infections. A two-step PCR based method amplifying a fragment of the 28S rRNA-ITS2 region was used to identify infections by Necator americanus and Ancylostoma spp. PCR products positive for Ancylostoma spp. were sequenced bidirectionally, and sequences analysed through BLAST and phylogenetic analysis. Samples containing Ancylostoma duodenale were further characterized by amplification and sequencing a fragment of cytochrome c oxidase subunit 1 (cox1) gene. PCR amplicons were successfully obtained from 42 (82.4%) of 51 samples, with 81.0% (34 of 42) identified as Necator americanus, 16.7% (7 of 42) as Ancylostoma spp. and 2.4% (1 of 42) as mixed infections of both species. All eight Ancylostoma spp. were confirmed to be Ancylostoma duodenale and this is the first time A. duodenale was reported in Malaysia. Samples containing A. duodenale from Nepalese and Indonesian workers shared high-similarity and were distinct compared to sequences from other countries. This study highlights the prevalence of hookworm infections among migrant workers living in Malaysia. Our findings underscore the necessity of screening migrant workers for hookworm infections, particularly those working in food-related services and industries.

Zoonotic Trichomonas tenax and a new trichomonad species, Trichomonas brixi n. sp., from the oral cavities of dogs and cats

Trichomonads are known to inhabit the oral cavities of various mammals, including dogs, cats and horses. However, little attention has been paid to species identification, prevalence and zoonotic potential of these parasites, although their hosts live in close proximity with humans. According to the original description, oral trichomonads in dogs and cats belong to the genus Tetratrichomonas. Interestingly, later investigations suggested that the oral cavities of dogs and cats could be infected with different species of the genus Trichomonas, including the human oral cavity parasite Trichomonas tenax. Thus, in this study we investigated the occurrence of oral trichomonads in 111 domestic dogs and 122 cats using cell cultivation methods, nested PCR analyses, and the sequencing of ITS1-5.8rRNA-ITS2 regions. We found that both dogs and cats harbour T. tenax, with prevalences of 8.1% and 4.1%, respectively. Considerably more dogs were infected with different species of the genus Trichomonas (30.6%), which we also identified in cats (6.6%). An analysis of the potential risk factors suggested that dogs of more than 3years old or with dental disease signs are more frequently infected with Trichomonas sp. than younger dogs or dogs without the disease signs, and that crossbreed dogs revealed increased rates of infection in comparison with purebred dogs. An analysis of the cat population suggested that Trichomonas sp. infection is lower in younger and crossbreed cats. Although the morphology of Trichomonas sp. is very similar to that of T. tenax, based on a phylogenetic analysis of ITS1-5.8rRNA-ITS2 regions and the ssrRNA genes, we consider Trichomonas sp. to represent a new trichomonad species, for which we propose the name Trichomonas brixi.