Internal Transcribed Sequence Research Articles

Morphological and Molecular Identification and Mating Type Detection of Chalkbrood Fungal Pathogen <i>Ascosphaera apis</i> in <i>Apis mellifera</i> L. in Southern India

Chalkbrood Ascosphaera apis is a fungal brood pathogen that exerts considerable biotic stress on honey bees worldwide. Chalkbrood was noticed at Coimbatore in Apis mellifera colonies for the first time. Precise identification of the fungal pathogen and their mating types is crucial to develop effective disease management strategies. Hence, the present study aimed to isolate and identify fungal pathogen morphologically and genomically. Scanning Electron Microscopic (SEM) analysis was done and confirmed the essential morphological characteristics of the isolated fungus such as spore cysts (51-86 µm in diameter), spore balls (8-15 µm in diameter), and ascospores (1.7-2.6 µm in length). Molecular characterization using internal transcribed sequence (ITS)-PCR of the fungal isolate indicated 99-100% sequence similarity to A. apis. In addition, the multiplex PCR assay was performed and the mating types MAT1-1 and MAT1-2 were successfully detected and named TNAU CBD MAT1 and TNAU CBD MAT2, respectively.

Epicoccum sp. as the causative agent of a reddish-brown spot defect on the surface of a hard cheese made of raw ewe milk

Colour defects can affect the appearance of cheese, its flavour, the safety of its consumption, and the price it can demand. This work reports the identification of five fungal isolates from a dairy plant where the surface of most cheeses was affected by patent, reddish-to-brown stains. One of these isolates was obtained from cheese, two from brine, and two from a bulk tank containing ewe milk. Molecular identification by partial amplification, sequencing, and database comparison of the concatenated sequence of the genes coding for the largest subunit of RNA polymerase II (RPB2), β-tubulin (β-TUB), and the large subunit of the rRNA molecule (LSU), plus the internal transcribed sequence (ITS) regions, assigned the isolates to Epicoccum layuense, Epicoccum italicum, and Epicoccum mezzettii. Features of the growth of these different species on different agar-based media, and of the morphology of their conidia following sporulation, are also reported. The strain isolated from cheese, E. layuense IPLA 35011, was able to recreate the reddish-brown stains on slices of Gouda-like cheese, which linked the fungus with the colour defect. In addition, two other strains, E. italicum IPLA 35013 from brine and E. italicum IPLA 35014 from milk, also produced stains on cheese slices. Epicoccum species are widely recognized as plant pathogens but have seldom been reported in the dairy setting, and never as human or animal pathogens.

Thermal Tolerance Data and Molecular Identification Are Useful for the Diagnosis, Control and Modeling of Diseases Caused by Thielaviopsis paradoxa.

Several economically important diseases of forest trees and agricultural crops in many parts of the world have been linked to the ascomycete fungal pathogen Thielaviopsis paradoxa. This study compared the growth rate of 41 isolates of T. paradoxa sourced from different hosts and two countries (Nigeria and Papua New Guinea (PNG)) under six temperature levels (22 °C, 25 °C, 30 °C, 32 °C, 34 °C and 35 °C). Phylogenetic relationships were obtained from the analysis of their nuclear ribosomal DNA internal transcribed sequence (ITS) data. While all the isolates from PNG and few from Nigeria grew optimally between 22 °C and 32 °C, the majority had their highest growth rate (2.9 cm/day) between 25 °C and 32 °C. Growth performances were generally low between 34 °C and 35 °C; no isolate from the sugar cane grew at these high temperatures. The oil palm isolate DA029 was the most resilient, with the highest growth rate (0.97 cm/day) at 35 °C. Phylogenetic analysis delineated five clusters: a very large clade which accommodates the majority (30 Nigerian and 3 PNG oil palm isolates) and four small clades containing two members each. To a large extent, the clustering pattern failed to address the temperature-isolate relationship observed. However, only the four small clades represent isolates with similar temperature tolerances. It is most likely that wider and robust analyses with more diverse isolates and genetic markers will provide better insight on thermal resilience of T. paradoxa. Additionally, future research to establish relationships between vegetative growth at different temperatures and of different pathogenicity and disease epidemiology merits being explored. The results might provide useful information for the formulation of effective management and control strategies against the pathogen, especially in this era of climate change.

Soil fungal diversity and assembly along a xeric stress gradient in the central Namib Desert

The Namib Desert of south-western Africa is one of the oldest deserts in the world and possesses unique geographical, biological and climatic features. While research through the last decade has generated a comprehensive survey of the prokaryotic communities in Namib Desert soils, little is yet known about the diversity and function of edaphic fungal communities, and even less of their responses to aridity. In this study, we have characterized soil fungal community diversity across the longitudinal xeric gradient across the Namib desert (for convenience, divided into the western fog zone, the central low-rainfall zone and the eastern high-rainfall zone), using internal transcribed sequence (ITS) metabarcoding. Ascomycota, Basidiomycota and Chytridiomycota consistently dominated the Namib Desert edaphic fungal communities and a core mycobiome composed of only 15 taxa, dominated by members of the class Dothideomycetes (Ascomycota), was identified. However, fungal community structures were significantly different in the fog, low-rainfall and high-rainfall zones. Furthermore, Namib Desert gravel plain fungal community assembly was driven by both deterministic and stochastic processes; the latter dominating in the all three xeric zones. We also present data that suggest that the inland limit of fog penetration represents an ecological barrier to fungal dispersal across the Namib Desert.

Yeast Biodiversity in Vineyard during Grape Ripening: Comparison between Culture Dependent and NGS Analysis

In this study, the evolution of the yeast microflora present on the berry surface, during the ripening of Barbera grapes, was monitored. Sampling was performed in three vineyards located in the “Nizza” Barbera d’Asti DOC zone and different methodologies have been employed. A culture-dependent method based on the identification of strains grown on solid media by ARDRA (Amplified Ribosomal DNA Restriction Analysis) and the D1-D2 domain of ribosomal 26S DNA capillary sequencing was coupled to NGS (Next Generation Sequencing) targeting ITS (Internal Transcribed Sequence) amplicons with the Illumina MiSeq platform. By using culture-dependent techniques, the most frequently detected species was the yeast-like fungus Aureobasidium pullulans, which was dominant in the culturable fraction. Among yeasts, the presence of oligotrophic basidiomycetes such as Cryptococcus spp., Rhodotorula graminis and Sporidiobolus pararoseus was observed at the beginning of ripening. Afterward, upon approaching the harvest, a succession of oxidative or weakly fermentative copiotrophic species occurs, such as Saturnispora diversa, Issatchenkia terricola, Hanseniaspora opuntiae, Starmerella bacillaris and Hanseniaspora uvarum. The massive sequencing revealed a larger number of species, respect to the culture-dependent data. Comparing the two different approaches used in this work, it is possible to highlight some similarities since Aureobasidium, Rhodotorula and Sporobolomyces were detected by both methods. On the contrary, genera Hanseniaspora, Issatchenkia and Saturnispora were revealed by culture-dependent methods, but not by NGS, while Saccharomyces spp. were identified, with low frequency, only by NGS. The integrated application of NGS sequencing and culture-dependent techniques provides a comprehensive view of mycodiversity in the wine-growing environment, especially for yeasts with low abundance.

Recombinase polymerase amplification combined with lateral flow dipstick for the rapid detection of Prorocentrum donghaiense

ABSTRACT Prorocentrum donghaiense is one of the most important harmful algal bloom (HAB)-forming species in the East China Sea. HABs caused by this alga has posed a considerable threat to marine ecosystems, fisheries, and human health. Therefore, exploring new methods for effectively detecting P. donghaiense is necessary. In this study, a novel method referred to as recombinase polymerase amplification combined with lateral flow dipstick (RPA−LFD), was developed for the detection of P. donghaiense. The internal transcribed sequence (ITS) of P. donghaiense was first obtained through PCR amplification, molecular cloning, and sequencing. Next, candidate RPA primers and LFD probes were designed through homologous sequence alignment analysis, and ITS was used as the target sequence. The optimal primers obtained through screening were used in establishing and optimizing the RPA−LFD detection system. Finally, the specificity of the system was verified, and its sensitivity and practicality were evaluated. Results showed that the RPA-LFD system had good specificity and did not cross-react with the other control algal species. The detection limit was 6.79 × 10−2 ng/μL for genomic DNA, 6.01 × 10 copies/μL for the ITS-containing recombinant plasmids, and 10 cells/mL for simulative water samples. The sensitivity of RPA−LFD was generally ten times that of conventional PCR. In conclusion, the proposed visual detection method for P. donghaiense is simple, rapid, sensitive, and applicable to field detection without complex instrumentation, and it is a promising tool for detecting P. donghaiense.

PHYLOGENETIC ASSESSMENT OF TREE SPECIES OF ACONITUM L. FROM KAZAKHSTAN BY USING ITS AND MATK MARKERS

The Aconitum L. is a diverse genus consisting of more than 300 species all over the World, including 11 species grown in Kazakhstan. The phylogeny of the genus was mostly studied by using internal transcribed sequences (ITS) of the nuclear genome and maturase K (matK) of the chloroplast genome. Therefore, in this study it was decided to assess the phylogenetic position of three local species A. leucostomum, A. soongoricum and A. apetalum, by using ITS and matK. The application of Maximum-Likelihood (ML) method suggested that the A. soongoricum belong to subgenus Aconitum, and A. leucostomum and A. apetalum to the subgenus Lycoctonum, which was congruent to previous taxanomic studies for this genus. The Median-Joining network using ITS suggested that A. sachalinense belongs to the group of processors of the genus among species that were involved in the analysis. The study is the first attempt to understand phylogenetic relationship of three species of Aconitum grown in Kazakhstan.

Short communication: Physicochemical features and microbial community of milk kefir using a potential probiotic Saccharomyces cerevisiae KU200284

The aim of this study was to analyze the β-glucan contents, physicochemical features, and microbial communities in milk kefir prepared using Saccharomyces cerevisiae KU200284 isolated from cucumber jangajji, a fermented vegetable commonly eaten in Korean. Three types of milk kefir were manufactured, with (1) activated kefir grain, (2) activated kefir grain with commercial S. cerevisiae BOF, and (3) activated kefir grain with S. cerevisiae KU200284. β-Glucan contents of milk kefir using kefir grain and kefir grain with S. cerevisiae strains BOF and KU200284 were 8.29, 8.59, and 8.57%, respectively. The pH, titratable acidity, viscosity, Brix level, and alcohol contents of milk kefir using kefir grain with S. cerevisiae strains were acceptable compared with milk kefir using only kefir grain. In milk kefir produced using kefir grains and S. cerevisiae strains, 16S rRNA reads showed representative strains of Lactobacillus kefiranofaciens (>72% relative abundance) and Acetobacter fabarum (>16% relative abundance). In particular, milk kefir using kefir grain with S. cerevisiae KU200284 had the highest relative abundance of L. kefiranofaciens. In addition, the internal transcribed sequence (ITS) rRNA reads in tested milk kefir showed representative strains of Kluyveromyces marxianus (>52% relative abundance) and Saccharomyces cerevisiae (>16% relative abundance). In contrast, milk kefir using S. cerevisiae strains had higher relative abundance of S. cerevisiae (>37%). The β-glucan production, physicochemical properties, and microbial community profiling indicate that S. cerevisiae KU200284 could be used in functional dairy products as a starter culture.

Morphological and molecular characterization of Macrophomina phaseolina isolated from three legume crops and evaluation of mungbean genotypes for resistance to dry root rot

Dry root rot (DRR) is an important emerging disease of mungbean caused by fungus Macrophomina phaseolina. The disease is seed and soil-borne and hence management is difficult. Exploitation of host resistance could be a good option to manage the disease. The objectives of this study were to characterize the isolates of M. phaseolina from three legume crops and to identify resistant sources against DRR of mungbean. Isolates of M. phaseolina from mungbean, urdbean, and vegetable soybean were identified using morphological characteristics and sequencing internal transcribed sequence (ITS) region of 18S rRNA. Isolates of M. phaseolina from urdbean and vegetable soybean were inoculated on susceptible check genotype of mungbean to assess their pathogenicity. Forty three mungbean genotypes were screened against M. phaseolina isolate of mungbean using the paper towel method to identify sources of resistance. Among these genotypes, 9 were resistant in repeated experiment, with disease score ranging from 1.9 to 3.0. Resistant genotypes showing lower disease score, namely IPM99-125, EC693368, and EC693369, were further screened using the sick pot method to confirm their resistance. Among these three genotypes, IPM99-125 showed consistently higher plant survival rate followed by EC693368 and EC693369 as compared to susceptible checks (VC3960-88, KPS1). In addition, these three genotypes were resistant when screened with two strains of M. phaseolina isolated from urdbean and vegetable soybean, with IPM99-125 having lower disease score. The mungbean genotype IPM99-125 could be useful in mungbean breeding programs to develop root rot-resistant varieties.

Đặc điểm hình thái và di truyền cây tầm vông (Thyrsostachys siamensis Gamble)

The study was carried out to examine ecological characteristics of bamboo (Thyrsostachys siamensis Gamble) cultivated in Luong Phi, Tri Ton, An Giang, and to analyze the morphological and genetic features. The results showed that total surveyed area including bamboos, perennial fruit trees, and timbers was 91.78 ha, whereas the bamboo area was 61.85 ha (67.39%). The soil analysis indicated that nutrients in bamboo cultivated region was poor. Regarding the morphology, most of the Luong Phi bamboos were cultivated for many years (above 15 years), so the rhizomes were lifted up. In term of anatomy, the external layer of Thyrsostachys siamensis Gamble is thicker than orther bamboos. Under every node, there is a white ring which is clearly observed (especially one season trees). The average diameters of culms at different positions from land surface to the shoot were 4.3, 3.5, and 1.5 cm. To understand the genetic characteristics of Thyrsostachys siamensis Gamble, the ITS (internal transcribed sequences) regions of Thyrsostachys siamensis Gamble and 3 other bamboos were sequenced. The nucleotide sequences were examined using BioEdit 7.0 software, then the sequences were aligned with ClustalW program. After that, the phylogenetic tree was constructed by Mega X software using bootstrap test with Maximum Likelihood algorithm. The result showed that Thyrsostachys siamensis Gamble had a close relationship with Denldrocalamus asper.

Temporal and Locational Variations of a Phytophthora spp. Community in an Urban Forested Water Drainage and Stream-Runoff System

Species richness and diversity of Phytophthora spp. (water molds) in urban riparian-forest ecosystems, which serve as primary drainage passageways for surface-water runoff, may be attributed to surrounding landscape management, associated vegetation, and environmental conditions. These riparian areas, although generally small, are always flooded during wet seasons and almost completely dry during the hottest parts of each year when there is limited precipitation. Little is known about Phytophthora spp. diversity within these heavily impacted sites. We sampled water, soil, and vegetation (phenology dependent) across 14 dates, over ~2 y at a site containing a drainage ditch that enters Hog Creek, in Rankin County, MS. We cultured all Phytophthora spp. using 4 published protocols to ensure maximum isolation potential. Across all sampling dates, 65 isolations were positive for Phytophthora spp., 12 of which were recovered from vegetation. We employed morphological and internal transcribed sequence (ITS) data to confirm taxa. We determined a total of 11 taxa on the basis of their phylogenetic clustering with known species of Phytophthora in a bayesian analysis. The most common taxa were P. chlamydospora, P. mississippiae, and P. cinnamomi at frequencies of 12.5%, 11.0%, and 10%, respectively. We verified morphologically and by sequence similarity an undescribed species, Phytophthora oaksoil taxon, which has been reported previously in the Western US, as well as other countries, such as Australia. Overall, the bottle-of-bait (BOB) intact-leaf and water-filtration methods had numerically greater frequencies (P ≤ 0.05) than BOB leaf disks, soil-baiting leaf disks, or vegetationsampling protocols. Overall frequency (14%) of Phytophthora spp. was significantly greater (P ≤ 0.05) for the 17 December 2014 sampling date. Even though several taxa identified in this study are reported to be pathogenic to riparian forest trees and vegetation at the Hog Creek site, symptoms on surrounding trees and vegetation was generally limited to foliar lesions, and we observed no visible damage or decline during the study period. It would be judicious to visit different, similar urban habitats to determine if common Phytophthora in this study are present in other central and southern Mississippi riparian habitats.

Internal Transcribed sequence (ITS) of Halocafeteria seosinensis (Bicosoecids)

The aim of the current study was to characterize and identify the smaller subunit (18S) and Internal Transcribed Spacer (ITS) regions, ITS1 and ITS2 (ITS1-5.8S-ITS2) of Halocafeteria seosinensis (Bicosoecida). Halocafeteria was isolated from the salterns of Morocco and maintained using standard techniques. The ITS1 and ITS2 are non-conserved regions which were amplified using universal primers, ITS1 and ITS4. The purified PCR products were directly sequenced by Sanger dideoxy sequencing technology and submitted to GenBank. For the construction of cladogram, closely related 18S rDNA sequences obtained from GenBank were multiple aligned using MEGA 6 software and the cladogram constructed by maximum likelihood method with 10,000 bootstrapping (BP) repetitions. The phylogenetic relationship among the other related stramenopiles were also studied. The molecular characterization by PCR of 18S genes was useful in confirming the taxonomic identity of a biflagellated H. seosinensis gen. et sp. nov. The ITS1-5.8S-ITS2 region of Halocafeteria is the one to be characterized for the very first time in the whole family of Bicosoecida. Due to discrepancy in placing Halocafeteria in Bicosoecida family, there is need in the characterization of ITS region sequences of stramenopiles and to use multigenes (such as 18S and ITS regions) for reconstructing the evolution tree.

Molecular Epidemiology of Ascaris Infection Among Pigs in Iowa.

Ascaris is a large roundworm parasite that infects humans and pigs throughout the world. Molecular markers have been used to study parasite transmission in Ascaris-endemic and -nonendemic regions of the world. In the United States, ascariasis still persists in commercial swine and has been designated a neglected disease of poverty in humans. However, relatively few data are available for evaluation of zoonotic transmission. In the present study, we obtained adult worms from abattoirs and characterized each worm on the basis of the gene encoding nuclear internal transcribed sequence (ITS) and mitochondrial cox1 Restriction fragment-length polymorphism analysis of ITS revealed swine, human, and hybrid genotypes. cox1 sequences were compared to all complete sequences available in GenBank, and haplotype analysis demonstrated 92 haplotypes worldwide. Sequences from the parasites in this study represented 10 haplotypes, including 6 new haplotypes that have not been previously described. Our results indicate that anthropozoonotic transmission has occurred in the past, resulting in the presence of human genotypes in pigs and supporting further investigation of zoonotic Ascaris transmission in the United States.

Pathogenic and molecular characterisation of <i>Pythium spp.</i> inducing root rot symptoms in other crops intercropped with beans in Southwestern Uganda

Objective: In Southwestern Uganda, bean root rot epidemics associated with Pythium species are frequent despite the use of various management methods. This study set out to determine whether other crops in bean cropping systems of Southwestern Uganda are affected by Pythium root rots and to characterise the Pythium species using Internal transcribed sequence (ITS) DNA primers. Methodology and Results: Root rots were found to occur on maize, sorghum, peas and potato sampled from farmer’s fields where they were found to be intercropped with beans affected by root rot. Pythium species were isolated using Corn meal agar (CMA).DNA was subsequently extracted and polymerase chain reaction ( PCR) analysis carried out using ITS DNA region primers and then the PCR products were sequenced. Twenty-one Pythium species were isolated. Cross pathogenicity tests were done in a screen house using bean pathogenic Pythium species and Pythium , species derived from other crops intercropped with beans. The Pythium species were moderately to non-pathogenic in maize and millet while those in sorghum and peas were highly pathogenic. Conclusion and application of results: This study found that other crops intercropped with beans in Southwestern Uganda were affected by Pythium root rots. Peas and sorghum were highly susceptible to the pathogenic Pythium species and could therefore be contributing to the continuous bean root rot epiphytotics in the region. On the other hand, maize and millet were non pathogenic to the Pythium species. This knowledge could be used to advice farmers not to include sorghum and peas when intercropping with beans, as they seem to be contributing to the Pythium inoculum load. They could alternatively use maize and millet, as they are not susceptible to the pathogenic Pythium species. Keywords: Pythium , root rots, Southwestern Uganda, ITS DNA, pathogenic

Root Rot of Chinese Jujube (Ziziphus jujuba) Caused by Cytospora sacculus in China.

Ziziphus jujuba Mill. (Rhamanaceae), commonly known as Chinese jujube, is a plant that is native to China and widely distributed in China and South Korea. Chinese jujube is a traditional Chinese medicine and food that has been used for thousands of years. From spring 2011 to spring 2012, Chinese jujube trees (<4 years old) from commercial orchards in Kashi and Akzo developed symptoms of severe wilting, chlorosis, and stunting. The taproot and lateral roots exhibited black, sunken, necrotic lesions, and progression of these symptoms eventually caused mortality of infected trees. In April 2011 and 2012, 15 trees with root rot symptoms were collected from different orchards, and a fungus was recovered consistently from symptomatic rhizome samples that had been surface-sterilized with 0.1% mercuric chloride and plated onto potato dextrose agar (PDA). Thirteen isolates were obtained. Colony growth on PDA was initially white but gradually turned grayish yellow-brown, with the lower side of the plate turning a deep yellow-brown. Colony texture was felty and slightly raised with no concentric growth zones. Pycnidia formed abundantly after 30 days, and each exuded a dull orange cirrhus containing conidia. The conidiomatal stromata were immersed in the bark, erumpent, discoid, convex to conical, and 1.53 ± 0.13 mm in diameter. Discs were dark brown or grey, nearly flat, circular, and up to 0.6 mm in diameter, with one to eight lateral ostioles. Ostioles were brown, 40 to 80 mm in diameter, and located above the disc surface. Locules were globose, with four to eight in a stroma, undivided, and did not share common walls. Conidia were hyaline, eguttulate, and allantoid, with sizes of 12 to 20 × 1.0 to 1.5 μm. The agar culture and conidiomatal stromata morphology were in accordance with descriptions of Cytospora sacculus (1,4). PCR amplification of the internal transcribed (ITS) spacer region of rDNA (using primers ITS1/4), combined with amplification of the β-tubulin gene (using primers BT1a/BT1b) (2,3), followed by sequencing of the amplified DNA, led to identification of isolate 6T-17 as Valsa ceratosperma (anamorph C. sacculus). The sequence of the ITS1-5.8s-ITS2 region of rDNA (GenBank Accession No. JX560175) matched the ITS sequence of V. ceratosperma Accession AB369475 with 99% similarity, and the β-tubulin gene sequence (KC840674) resulted in a 99% match with that of V. ceratosperma Accession EU219136. Mycelial plugs of a culture of each fungal isolate on PDA were inoculated onto 10 rhizomes of 2-month-old seedlings of Z. jujube, and maintained in a greenhouse at 25 to 30°C. Ten rhizomes of 2-month-old seedlings of Z. jujuba were inoculated with non-colonized PDA plugs as the control treatment. After 2 months, all inoculated seedlings showed chlorotic leaves and root rot symptoms similar to those observed in the original commercial orchards. No symptoms were observed on the control plants. The pathogen was reisolated successfully from symptomatic plants but not from the control plants, fulfilling Koch's postulates. To our knowledge, this is the first report of C. sacculus causing a root rot of Z. jujuba trees in China.

Lymnaea neotropica and Lymnaea viatrix, potential intermediate hosts for Fascioloides magna

Experimental infections of two South American lymnaeid populations with Fascioloides magna were carried out to determine whether these snails may sustain larval development of this digenean and, if so, to quantify their potential for cercarial production. The reference group was a French population of Galba truncatula infected and raised according to the same protocol. According to the internal transcribed sequence (ITS)-1 segment of their genomic rDNA, these South American populations were identified as Lymnaea neotropica (origin, Argentina) and Lymnaea viatrix var. ventricosa (origin, Uruguay). In the snail groups followed for cercarial shedding, longer prepatent periods and lower numbers of shed cercariae were noted in South American lymnaeids. In other snails dissected at day 65 post-exposure, the redial and cercarial burdens of F. magna found in the bodies of L. neotropica and L. v. ventricosa were significantly lower than those noted in G. truncatula. Compared to the total cercarial production noted in the dissected snails, the percentage of cercariae that exited from snails was 51.3% for G. truncatula, 32.2% for L. neotropica and 46.8% for L. v. ventricosa. The two South American species of snails can thus be considered as potential intermediate hosts of F. magna.

Identification and Molecular Analysis of Pathogenic Yeasts in Droppings of Domestic Pigeons in Beijing, China

Feral pigeons are known as reservoirs of pathogenic yeasts that cause opportunistic infections in human. In the outskirts of Beijing, China, pigeons are more frequently raised at homes than are encountered in public areas. Many studies have focused on the presence of pathogenic yeasts in the excreta (fresh or withered) of a variety kinds of birds, pigeon crop and cloacae. One hundred and forty-three samples of fresh droppings were collected from three suburban pigeon-raising homes in an area of northern Beijing, China. The internal transcribed sequences (ITS) of all strains (except for 8 strains of Rhodotorula sp. ) were sequenced and compared with those of the databases of the National Center for Biotechnology Information website ( http://www.ncbi.nlm.nih.gov ) using the Basic Local Alignment Search Tool (BLAST). Yeasts representing 8 genera, Cryptococcus, Filobasidium, Rhodotorula, Candida, Debaryomyces, Saccaromyces, Trichosporon and Sporidiobolus, were identified from 120 isolates. Cryptococcus was the most prolific genera represented by eight species. The populations of yeast species isolated from fresh pigeon droppings were different among homes. Although it is well established that Cryptococcus neoformans exists mainly in old pigeon guano, several C. neoformans strains were still isolated from fresh pigeon excreta, providing a clue that live cryptococcal cells could move through the gastrointestinal tract of the pigeons. Eight genera identified from fresh droppings of domestic pigeons further confirm that pigeons serve as reservoirs, carriers and even spreaders of Cryptococcus species and other medically significant yeasts. The proportion of pathogenic yeasts in all isolates is more than 90 %.

Chemiluminescent DNA optical fibre sensor for Brettanomyces bruxellensis detection

Food and beverage industries require rapid tests to limit economic losses and one way to do so is via molecular tests. In the present work, DNA capture and secondary probes, were designed to target the ITS (Internal Transcribed) sequences of Brettanomyces bruxellensis, a yeast responsible for the production of off flavours in both wine and beer. ITS1 and ITS2 were found to contain distinct regions with sufficient sequence divergence to make them suitable as specific identification target sites. The dot blot technique was used to determine the sensitivity and specificity of the capture probe. Both probes were, thereafter, adapted to construct an optical fibre genosensor, which produced neither false positives nor false negatives, and was both repeatable and faster with respect to traditional methods, the latter requiring at least one week to detect B. bruxellensis.

Multiple occupancy of nodules by nodulating rhizobia on field-grown soybeans with attendance of Sinorhizobium spp.

This study analyzed the phenotypic and genotypic characters of nodulating rhizobia isolated from two soybean cultivars, Kyushu 151 and Sachiyutaka, in the same field of the Yamaguchi Prefectural Technology Center of Agriculture and Forestry in Japan. The isolates were classified into groups using phenotypic characteristics, such as growth rate, color change on Bromothymol blue-containing yeast extract-mannitol agar (YMA) plates and colony morphology on YMA plates, and by genotypic characteristics, such as polymerase chain reaction–restriction fragment length polymorphism patterns of the 16S ribosomal RNA genes (16S rDNA) and the internal transcribed sequence (ITS) regions. In Kyushu 151, single phenotypic and genotypic groups were isolated from every nodule examined. In Sachiyutaka, plural strains belonging to distinct groups were obtained frequently from single nodules, indicating that multiple occupancy was established at high frequency. No fixed combination of the groups was found in the composition of multiple occupancy. An increase in the relative abundance of isolates belonging to Sinorhizobium fredii (Ensifer fredii) occurred concomitantly with the increase in the proportion of nodules with multiple occupancy. Nearly 60% of the isolates from Sachiyutaka belonged to S. fredii; 75% of them were obtained from nodules with multiple occupancy.

Mycobacterium chelonae valve endocarditis resulting from contaminated biological prostheses

A rapid-growing mycobacteria biological prosthetic valve (BPV) endocarditis related to prosthetic manufacturing process is described in Brazil. From 1999 to 2008, thirty-nine patients underwent BPV replacement due to culture-negative suspected endocarditis. All these cases had histological sections stained by Ziehl-Neelsen method. Clinical and microbiological data were reviewed in all acid-fast bacilli (AFB) positive cases. The 16S-23S internal transcribed sequence (ITS) was amplified using DNA extracted from paraffin-embedded samples, digested with restrictions enzymes and/or sequenced. Eighteen AFB positive BPV (18/39)(46%) were implanted in 13 patients and were from the same manufacturer. Four of them were implanted in other hospitals. Thirteen BPV were histologically proven endocarditis and five showed a colonization pattern. The examination of six non-implanted "sterile" BPV from this manufacturer resulted in 5 AFB positive. Mycobacterium chelonae was the AFB identified by ITS restriction analysis and sequencing. Rapid-growing mycobacteria infections must be suspected and Ziehl-Neelsen stain always performed on histology of either early or late BPV endocarditis, particularly when blood cultures are negative.