Internal Transcribed Spacer rDNA Sequences Research Articles

The Use of DNA Barcoding and Phylogenetic Analysis to Improve Identification of <i>Usnea</i> spp. Based on ITS rDNA

Lichen of the genus Usnea is quite common being used as a traditional herbal remedy. This genus is characterized by thallus, which is very similar among the species, leads to some difficulties in distinguishing them. In Indonesia, such research report on the availability of this genus based on their morphological characteristic is minimal. This might be due to too high morphological similarities among them. The molecular character, which is based on the DNA Barcode of Internal Transcribed Spacer (ITS) rDNA sequences, with its conserved region (5.8S) and varied region (ITS1 and ITS2), are becoming essential characters on identifying as well as analyzing the phylogenetic. The current study then proposed to identify and draw the species dendrogram of species within the Usnea genus obtained from Mount Lawu Forest of Central Java and Turgo Forest of Yogyakarta based on their phylogenetic and phenetic analysis. The dendrogram was constructed with UPGMA using the simple matching coefficient, whereas the phylogenetic tree was constructed with Maximum Likelihood (ML) using Kimura-2 parameter with 1000 bootstrap. The data were unable to draw phenetic relationships among the subgenus Usnea and Eumitria members. The phylogenetic tree shows the primary two clades, distinguishing the subgenus Usnea and Eumitria. The ITS rDNA sequence was able to identify most of the Usnea species.

Airway bacterial and fungal microbiome in chronic obstructive pulmonary disease

Little is known about airway mycobiome, and its relationship with bacterial microbiome in chronic obstructive pulmonary disease (COPD). Here we report the first simultaneous characterization of sputum bacterial and fungal microbiome in 84 stable COPD and 29 healthy subjects, using 16S ribosomal DNA and fungal internal transcribed spacer DNA sequencing. Ascomycota predominated over Basidiomycota in fungal microbiome both in COPD patients and healthy controls. Meyerozyma, Candida, Aspergillus and Schizophyllum were most abundant at the genus level. There was a significant inverse correlation between bacterial and fungal microbial diversity, both of which altered in opposite directions in COPD patients versus controls, and in frequent versus non-frequent exacerbators. An enhanced bacterial-fungal ecological interaction was observed in COPD patients, which was characterized by higher proportion of co-occurrence intrakingdom interactions and co-exclusive interkingdom interactions. In COPD, four mutually co-occurring fungal operational taxonomic units (OTUs) in Candida palmioleophila, Aspergillus and Sordariomycetes exhibited co-exclusive relationships with other fungal OTUs, which was specifically present in frequent exacerbators but not in non-frequent exacerbators. The perturbed bacterial-fungal interactions in COPD were associated with increased airway inflammatory mediators such as IL-6 and IL-8. The disruption of airway bacterial-fungal community balance, characterized by the loss of commensal bacterial taxa and enriched pathogenic fungal taxa, is implicated in COPD. The emergence of pathogenic fungi such as Candida and Aspergillus could be a marker for the frequent exacerbator phenotype. The airway mycobiome is an important cofactor mediating pathogenic infection and host inflammation in COPD.

Isolation, identification and genotyping of Candida albicans from Landes geese

In May 2018, Landes geese raised in Weifang, Shandong Province, China, developed a disease characterized by thickened oesophageal mucosa and white, round ulcers. Based on pathogen isolation and identification, differential culture and morphological observations, Candida albicans (C. albicans) was identified as the causative pathogen from the oesophagus of infected geese, and artificial infection experiments were then performed using the isolated strains. In experimental reproduction, the symptoms of infected geese were consistent with those of natural infection, and gosling morbidity and mortality were 75% and 60%, respectively. Re-isolation of the strain from the dead goslings confirmed C. albicans as the causative pathogen of oesophageal ulcers. We further performed internal transcribed space rDNA sequence analysis, ABC genotyping and multi-locus sequence typing analysis. We observed 100% sequence similarity between the two strains, designated as WFCL and WFLQ, which were isolated from different regions, with 100% homology between the strains isolated in the present study and the human-origin C. albicans strains isolated previously from China. The goose-origin strains isolated in this study and the human-origin C. albicans isolates were included in the same branch in phylogenetic trees analysis, indicating that the strain responsible for oesophageal ulcer in geese is closely related to human-origin C. albicans. In addition, based on eBURST analysis of sequence types, goose-origin C. albicans strains were relatively independent in terms of population evolution. To the best of our knowledge, this is the first detailed report on goose oesophageal ulceration caused by C. albicans infection in geese. Considering that C. albicans is an important zoonotic pathogen, this study provides a reference for further studies on avian C. albicans infections and is important for ensuring public health and safety.

Evolution of Tandem Repeats Is Mirroring Post-polyploid Cladogenesis in Heliophila (Brassicaceae).

The unigeneric tribe Heliophileae encompassing more than 100 Heliophila species is morphologically the most diverse Brassicaceae lineage. The tribe is endemic to southern Africa, confined chiefly to the southwestern South Africa, home of two biodiversity hotspots (Cape Floristic Region and Succulent Karoo). The monospecific Chamira (C. circaeoides), the only crucifer species with persistent cotyledons, is traditionally retrieved as the closest relative of Heliophileae. Our transcriptome analysis revealed a whole-genome duplication (WGD) ∼26.15–29.20 million years ago, presumably preceding the Chamira/Heliophila split. The WGD was then followed by genome-wide diploidization, species radiations, and cladogenesis in Heliophila. The expanded phylogeny based on nuclear ribosomal DNA internal transcribed spacer (ITS) uncovered four major infrageneric clades (A–D) in Heliophila and corroborated the sister relationship between Chamira and Heliophila. Herein, we analyzed how the diploidization process impacted the evolution of repetitive sequences through low-coverage whole-genome sequencing of 15 Heliophila species, representing the four clades, and Chamira. Despite the firmly established infrageneric cladogenesis and different ecological life histories (four perennials vs. 11 annual species), repeatome analysis showed overall comparable evolution of genome sizes (288–484 Mb) and repeat content (25.04–38.90%) across Heliophila species and clades. Among Heliophila species, long terminal repeat (LTR) retrotransposons were the predominant components of the analyzed genomes (11.51–22.42%), whereas tandem repeats had lower abundances (1.03–12.10%). In Chamira, the tandem repeat content (17.92%, 16 diverse tandem repeats) equals the abundance of LTR retrotransposons (16.69%). Among the 108 tandem repeats identified in Heliophila, only 16 repeats were found to be shared among two or more species; no tandem repeats were shared by Chamira and Heliophila genomes. Six “relic” tandem repeats were shared between any two different Heliophila clades by a common descent. Four and six clade-specific repeats shared among clade A and C species, respectively, support the monophyly of these two clades. Three repeats shared by all clade A species corroborate the recent diversification of this clade revealed by plastome-based molecular dating. Phylogenetic analysis based on repeat sequence similarities separated the Heliophila species to three clades [A, C, and (B+D)], mirroring the post-polyploid cladogenesis in Heliophila inferred from rDNA ITS and plastome sequences.

Optimization of In Vitro Culture Conditions for Production of Cordyceps bassiana Spores (Ascomycetes) and the Effect of Spore Extracts on the Health of Caenorhabditis elegans.

Cordyceps bassiana, an entomogenous fungus, has been used as a dietary therapeutic in traditional Chinese medicine for several millennia. However, naturally occurring C. bassiana is notably scarce in the field, especially at the spore stage. To date, there are few comprehensive studies on the biological characteristics and pharmacological attributes of C. bassiana spores. In this study, an isolate of C. bassiana (Bs1112) was identified as the teleomorph of Beauveria bassiana using morphology and rDNA internal transcribed spacer sequences. In vitro solid substrate fermentation parameters were evaluated; a rice substrate with 0.01% K+ achieved the maximum spore yield. High-performance liquid chromatography analysis of the bioactive components of C. bassiana spore powder (BC-CBSP), which was extracted using alcohol, showed that concentrations of adenosine were higher than concentrations of inosine, uridine, and cordycepin. The pharmacological impact of BC-CBSP was elucidated in a live animal model, Caenorhabditis elegans, at a range of concentrations from 0.145 to 14.5 mg/mL. The reproductive rate, frequency of head swinging and body bending (indicative of C. elegans locomotion), and lifespan of C. elegans were determined and compared with untreated populations. At the lowest concentration of BC-CBSP, reproduction was slowed but the number of eggs was not reduced. However, at both of the higher concentrations (1.45 and 14.5 mg/mL), BC-CBSP reduced the total number of eggs produced during the 8-day observation period. Furthermore, 0.145 mg/mL of BC-CBSP significantly increased the frequency of head swinging/body bending and the lifespan of C. elegans. This is the first report on the pharmacological effects of C. bassiana spores. Results suggest that low concentrations of BC-CBSP have no negative effects on the reproduction rate of C. elegans but could enhance nervous system activity and extend their lifespan.

A global meta-analysis of ITS rDNA sequences from material belonging to the genus Ganoderma (Basidiomycota, Polyporales) including new data from selected taxa.

Ganoderma P. Karst. is a cosmopolitan genus of white-rot fungi which comprises species with highly-prized pharmaceutical properties, valuable biotechnological applications and of significant phytopathological interest. However, the status of the taxonomy within the genus is still highly controversial and ambiguous despite the progress made through molecular approaches. A metadata analysis of 3908 nuclear ribosomal internal transcribed spacer (ITS) rDNA sequences obtained from GenBank/ENA/DDBJ and UNITE was performed by targeting sequences annotated as Ganoderma, but also sequences from environmental samples and from material examined for the first time. Ganoderma taxa segregated into five main lineages (Clades A to E). Clade A corresponds to the core of laccate species and includes G. shanxiense and three major well-supported clusters: Cluster A.1 (‘G. lucidum sensu lato’) consists of taxa from Eurasia and North America, Cluster A.2 of material with worldwide occurrence including G. resinaceum and Cluster A.3 is composed of species originating from all continents except Europe and comprises G. lingzhi. Clade B includes G. applanatum and allied species with a Holarctic distribution. Clade C comprises taxa from Asia and Africa only. Clade D consists of laccate taxa with tropical/subtropical occurrence, while clade E harbours the highest number of non-laccate species with a cosmopolitan distribution. The 92 Ganoderma-associated names, initially used for sequences labelling, correspond to at least 80 taxa. Amongst them, 21 constitute putatively new phylospecies after our application of criteria relevant to the robustness/support of the terminal clades, intra- and interspecific genetic divergence and available biogeographic data. Moreover, several other groups or individual sequences seem to represent distinct taxonomic entities and merit further investigation. A particularly large number of the public sequences was revealed to be insufficiently and/or incorrectly identified, for example, 87% and 78% of entries labelled as G. australe and G. lucidum, respectively. In general, ITS demonstrated high efficacy in resolving relationships amongst most of the Ganoderma taxa; however, it was not equally useful at elucidating species barriers across the entire genus and such cases are outlined. Furthermore, we draw conclusions on biogeography by evaluating species occurrence on a global scale in conjunction with phylogenetic structure/patterns. The sequence variability assessed in ITS spacers could be further exploited for diagnostic purposes.

The mycorrhizal community of the epiphytic orchid Thrixspermum japonicum is strongly biased toward a single Ceratobasidiaceae fungus, despite a wide range of fungal partners.

Orchids depend primarily on mycorrhizal fungi to obtain nutrients throughout their life cycle. Epiphytic orchids account for 69% of orchid diversity. The unstable availability of water and nutrients in their arboreal habitats often results in severe water and nutrient stresses. Consequently, mycorrhizal associations may be important for the survival of epiphytic orchids, but our understanding thereof remains limited. Here, we investigated the mycorrhizal community in a single epiphytic orchid species, using more samples than in any previous study. We assessed the mycorrhizal communities of Thrixspermum japonicum, one of the most common epiphytic orchids in the temperate region of Japan. In total, 144 individuals were collected from 28 host tree species at 20 sites across 1300km. The mycorrhizal fungi were identified based on nuclear ribosomal DNA internal transcribed spacer sequences and assigned operational taxonomic units (OTUs) based on 97% sequence similarity. We obtained 24 OTUs; 9 belonged to the Ceratobasidiaceae and 15 to the Tulasnellaceae. These OTUs are widely distributed throughout the phylogenetic trees of the two fungal families. However, a single Ceratobasidiaceae OTU accounted for 49.7% of all fungal sequences and was predominant in samples from 15 host tree species and 12 sites. Our results imply that despite having a broad range of mycorrhizal partners, T. japonicum was predominantly associated with a single fungal taxon at most of the sites among the host-tree species investigated. These findings contribute to elucidating mycorrhizal symbiosis in epiphytic habitats.

Morphological and molecular characterization of Podosphaera lini on Linum usitatissimum in IRAN

Linum usitatissimum, known as flax, is grown all around the world. Powdery mildew is a common disease on this crop so that this disease has been described from most flax-growing areas of the world. Clear symptoms of powdery mildew were observed on the field of different flax cultivars of Seed and Plant Improvement Institute of Iran in 2020 growing season. The causal pathogen of flax powdery mildew was identified as Podosphaera lini based on the morphological features and the nuclear ribosomal DNA internal transcribed spacer sequence. This is the first documented report of P. lini from flax in Iran.

Pathological, biochemical and molecular variability of Colletotrichum truncatum incitant of anthracnose disease in chilli (Capsicum annuum L.)

The present study aims to establish pathogenic variability among Colletotrichum truncatum, an incitant of anthracnose disease across different chilli growing regions of Karnataka. Thirty suspected C. truncatum isolates were identified based on their morphological and conidial characteristics and further confirmed by Internal Transcribed Spacer DNA sequence analysis. Pathogenicity test was carried out by in vitro detached leaf and fruit assay, and also under greenhouse conditions using 20 different chilli cultivars grown across Karnataka. Colletotrichum truncatum isolates recorded the varied degree of pathogenicity index (PI) on different chilli cultivars. Isolate UOM-02 was found highly virulent (PI > 80 against 12 tested cultivars) and cultivar cv. 4 was found highly resistant to C. truncatum infection (Average PI, 48.21). Further, the involvement of enzymes such as cellulase, pectin methylesterase and ascorbate peroxidase in determining the virulence of the pathogen was established. The highest activity of catalase (UOM-24; 7.38 units), ascorbate peroxidase (UOM-02; 2.9 units), cellulase (UOM-02; 0.58 units), and pectin methylesterase (UOM-02; 6.7 units), was recorded by different C. truncatum isolates. Cellulase and pectin methylesterase activities were positively correlated with their pathogenicity, while catalase activity was found least correlated. Results of RAPD and ISSR analysis recorded higher polymorphism among the isolates. Interestingly these isolates were not clustered based on their geographical origin, Pathogenicity index and biochemical characters. From this study, the existence of highly virulent C. truncatum isolate (UOM-02), which can cause severe loss under favourable conditions, was revealed. Further, possible use of specific enzymes as an indicator of virulence of the pathogen is discussed.

First Report of Powdery Mildew Caused by Erysiphe viciae-unijugae on Vicia sativa subsp. nigra in Korea.

Vicia sativa subsp. nigra, also known as narrow-leaved vetch, belongs to the family Fabaceae and is distributed in different countries.Vetch is one of the most important annual forage legumes in the world; it is used as hay, grain, silage, green manure, and livestock fodder. In Korea, this plant is often used as cattle feed. In early May of 2019 and 2020, a powdery mildew was observed on leaves of V. sativa subsp. nigra plants in a garden of Chonnam National University, Gwangju, Korea. The first symptom appeared on the upper surface of the leaves as a small white spot. Within two weeks, the white spots of powdery mildew spread and covered the entire leaf. Voucher specimens were deposited at the Environmental Microbiology Laboratory Herbarium, Chonnam National University, Gwangju, Korea, under the accession numbers CNUFC PWV1 and CNUFC PWV2. The hyphal appressoria of the fungus were nipple-shaped, moderately lobed or multilobed, single or in opposite pairs. Measurements were made of 35 conidiophores and 55 conidia. Conidiophores arising from the upper part of mother cells, were cylindrical, 60.5 to 117.5 × 7.5 to 9.5 μm. The foot cells of the conidiophores were cylindrical, straight to slightly curved at the base, 23.5 to 38.7 × 7.3 to 9.2 μm, followed by 2-3 shorter cells. Conidia were formed singly, subcylindrical to oval, 32.5 to 48.7 × 13.5 to 19.7 μm. Conidia produced short to long germ tubes, terminating in lobed or unlobed appressoria. No chasmothecia were observed. The morphological characteristics of conidiophores and conidia were consistent with the description of Erysiphe viciae-unijugae in Shin (2000). However, it was not possible to identify the fungus only based on the asexual morph. In addition, the conidiophore and conidium shapes of E. baeumleri on the known host V. sativa were similar to those of E. viciae-unijugae (Braun and Cook 2012). Therefore, genomic DNA was extracted directly from the mycelia and conidia using a DNA prep kit (Solgent, Daejeon, Korea). The internal transcribed spacer (ITS) regions ofribosomal DNA (rDNA) and 28S rDNA from the two specimens were amplified using the primers ITS5 and PM6 (Takamatsu and Kano 2001), and LROR and LR3 (Vilgalys and Hester 1990), respectively. A BLASTn search with the ITS sequences indicated that CNUFC PWV1 (GenBank accession no. MT509395) and CNUFC PWV2 (MT509396) were 100% (503/503 bp) and 99.8% (502/503 bp) identical to those of E. viciae-unijugae (LC010086 and LC009962), respectively. The 28S sequences of CNUFC PWV1 (MT705629) and CNUFC PWV2 (MT705630) showed 99.8% (507/508 bp) and 99.4% (505/508 bp) identity values with E. viciae-unijugae (LC009976 and LC010029), respectively. Phylogenetic analyses based on the combined ITS and 28S rDNA sequences indicated that the two specimens, CNUFC PWV1 and CNUFC PWV2, formed a monophyletic clade together with sequences retrieved from E. viciae-unijugae. Therefore, the causal pathogen was identified as E. viciae-unijugae based on the results of morphological and molecular analyses. Pathogenicity tests were performed by gently pressing the infected leaves onto 10 young leaves of three healthy plants. A non-inoculated plant was used as a control. The plants were maintained in a greenhouse at 22 to 25°C, with relative humidity around 75%, and natural light. Powdery mildew symptoms were observed on the inoculated leaves after 10-14 days, whereas no symptoms were observed on the leaves of the control. The fungi on the inoculated leaves were morphologically identical to the fungi on the original infected leaves. Erysiphe viciae-unijugae has been reported to occur on Lathyrus spp. and Vicia spp. (Farr and Rossman 2020). To our knowledge, this is the first report of powdery mildew caused by E. viciae-unijugae on V. sativa subsp. nigra in Korea. The weather conditions, especially during May, are favorable for disease outbreaks. This disease may be a potential threat to feed production in Korea.

Bioremediation of Crude Oil by Rhizosphere Fungal Isolates in the Presence of Silver Nanoparticles.

Background: This research work focuses on the utilization of indigenous fungi for in situ bioremediation of crude oil in the presence of silver nanoparticles. Methods: Two fungi belonging to two different genera showed promising crude oil-degrading abilities. Fungal isolates were identified based on internal transcribed spacer rDNA sequence analysis. Gas chromatography-mass spectrometry analysis of the crude oil remaining in the culture medium after seven days was performed. The response surface method (RSM) designed by Box-Behnken was used to establish a mathematical model. Inter-simple sequence repeat (ISSR) primers were used to examine the genetic variation of fungal isolates. Results: Gas chromatography-mass spectrometry (GC-MS) analysis after seven days showed that the optimum biodegradation of crude oil was 57.8%. The crude oil degradation rate was significantly affected by a temperature of 30 °C, pH value of 7, crude oil concentration of 4 g/L, a 1:1 ratio between A. flavus AF15 and T. harzianum TH07, and an silver nanoparticle (AgNP) concentration of 0.05 g. Molecular characterization in fungal isolates is extremely valuable when using ISSR markers. Conclusions: Two fungal isolates showed promising crude oil-degrading abilities with positive effect of low concentrations of AgNPs on biodegradation. RSM is an efficient mathematical method to optimize the microbial biodegradation of crude oil.

A Molecular Phylogenetic Study of Clarohilum henningsii (Mycosphaerellaceae, Fungi) on Cassava from Indonesia based on the ITS rDNA Sequence

Leaf spot is a common disease of cassava in Indonesia and other tropical countries. The present study aims to determine two isolates isolated from leaf spot of cassava in Indonesia using a molecular phylogenetic analysis based on internal transcribed spacer (ITS) rDNA sequence and morphological examination. The results showed that the two isolates belong to Clarohilum henningsii. This study revealed the phylogenetic position of C. henningsii from Indonesia, where in the previous studies, it has been reported as Cercospora cassavae, C. manihotis, C. henningsii, or Passalora henningsii based on morphological examinations.

First Report of Colletotrichum salicis Causing Bitter Rot of Apple in Italy.

Apple (Malus domestica Borkh.) is the most important fruit crop in South Tyrol (northern Italy), with a total production of 905.089 tons in 2019 (Chamber of Commerce, Industry, Crafts and Agriculture of Bozen-Bolzano 2020). Symptoms of bitter rot were observed on organic apples of the cultivar 'Roho 3615'/Evelina® collected in a packinghouse in South Tyrol at the end of March 2018 after approximately six months of storage in controlled atmosphere. Lesions were circular and light brown with orange conidial masses. Tissue samples were removed under aseptic conditions from surface-cleaned fruit at the margin between healthy and diseased pulp tissue, transferred to Petri dishes with potato dextrose agar (PDA), and incubated in the dark at room temperature for two weeks. Single spore cultures were obtained by adapting the procedure of Choi et al. (1999). Pure cultures were grown in quadruplicate on PDA at 20°C in the dark for two weeks. The colony appearance on the upper side was mostly flat with a distinct margin, the surface was covered with short, floccose aerial mycelium, and the color ranged from light gray to dark gray, while the reverse side appeared yellow. From each replicate culture, conidia were harvested, and the length and width of 50 randomly selected conidia were measured using a compound light microscope coupled to a digital camera (Leica DMLS, Leica Microsystems, Wetzlar, Germany). Conidia were cylindrical to fusiform, pointed at one end, and measured 10.0 to 19.5 × 2.5 to 5.0 μm (14.5 ± 1.9 × 3.9 ± 0.7 μm [mean ± SD]) in consistency with Damm et al. (2012). In order to determine the species of isolate 18-DSS-BS-EL-1-012, a multi-locus DNA sequence analysis was performed. Genomic DNA was extracted by following the protocol described by Cassago et al. (2002). Four loci, actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (HIS3) and the internal transcribed spacer (ITS) region of the rRNA operon were amplified by PCR and Sanger sequenced (Damm et al. 2012; Lévesque and de Cock 2004). The obtained DNA sequences of ACT, GAPDH, HIS3 and ITS were 186, 150, 317 and 505 bp long and were submitted to GenBank under the accession numbers MT347599, MT347600, MT347598 and MT337388, respectively. A MegaBLAST analysis resulted in 100% sequence identity at all four loci with a type culture of Colletotrichum salicis (CBS 607.94; GenBank accession numbers: JQ949781, JQ948791, JQ949451 and JQ948460), which belongs to the C. acutatum species complex (Damm et al. 2012). A pathogenicity test was performed with twelve 'Golden Delicious' apples by wounding the fruit with a sterile piercing tool and inoculating 20 μl of spore suspension (104 conidia per ml) from a 21-day-old PDA culture. Inoculated fruits were incubated for 21 days in a moist chamber at 20°C in the dark. The symptoms were recorded at 3, 5, 7, 10, 14 and 21 days post-inoculation (dpi). Symptoms appeared after 7 days on all inoculated fruits and resembled those observed on the original fruit, while mock-inoculated controls with sterile water remained symptomless. Fungal colonies resembling the original culture were re-isolated from lesions on the apple and plated on PDA. Their identity was confirmed by DNA sequence analysis of the ITS region, thereby proving Koch's postulates. Bitter rot occurs globally and is considered one of the most important diseases of apple that has the potential to cause significant crop losses (Sutton et al. 2014). Other Colletotrichum species have been commonly reported from apple in Europe, such as C. godetiae and C. fioriniae, whereas C. salicis has been reported solely in New Zealand and Belgium (Damm et al. 2012; Grammen et al. 2019). To the best of our knowledge, this is the first report of C. salicis causing bitter rot of apple in Italy.

Molecular phylogeny and species delimitation of Stachyuraceae: Advocating a herbarium specimen‐based phylogenomic approach in resolving species boundaries

AbstractSpecies concept and delimitation are fundamental to taxonomic and evolutionary studies. Both inadequate informative sites in the molecular data and limited taxon sampling have often led to poor phylogenetic resolution and incorrect species delineation. Recently, the whole chloroplast genome sequences from extensive herbarium specimen samples have been shown to be effective to amend the problem. Stachyuraceae are a small family consisting of only one genus Stachyurus of six to 16 species. However, species delimitation in Stachyurus has been highly controversial because of few and generally unstable morphological characters used for classification. In this study, we sampled 69 individuals of seven species (each with at least three individuals) covering the entire taxonomic diversity, geographic range, and morphological variation of Stachyurus from herbarium specimens for genome‐wide plastid gene sequencing to address species delineation in the genus. We obtained high‐quality DNAs from specimens using a recently developed DNA reconstruction technique. We first assembled four whole chloroplast genome sequences. Based on the chloroplast genome and one nuclear ribosomal DNA sequence of Stachyurus, we designed primers for multiplex polymerase chain reaction and high throughput sequencing of 44 plastid loci for species of Stachyurus. Data of these chloroplast DNA and nuclear ribosomal DNA internal transcribed spacer sequences were used for phylogenetic analyses. The phylogenetic results showed that the Japanese species Stachyurus praecox Siebold & Zucc. was sister to the rest in mainland China, which indicated a typical Sino‐Japanese distribution pattern. Based on diagnostic morphological characters, distinct distributional range, and monophyly of each clade, we redefined seven species for Stachyurus following an integrative species concept, and revised the taxonomy of the family based on previous reports and specimens, in particular the type specimens. Furthermore, our divergence time estimation results suggested that Stachyuraceae split from its sister group Crossosomataceae from the New World at ca. 54.29 Mya, but extant species of Stachyuraceae started their diversification only recently at ca. 6.85 Mya. Diversification time of Stachyurus in mainland China was estimated to be ca. 4.45 Mya. This research has provided an example of using the herbarium specimen‐based phylogenomic approach in resolving species boundaries in a taxonomically difficult genus.

Screening and molecular characterization of new thermo- and ethanol-tolerant Acetobacter malorum strains isolated from two biomes Moroccan cactus fruits.

Industrially, the sensitivity of acetic acid bacteria (AAB) to the high temperatures and the high ethanol concentrations is the major concerns for manufacturers. This study was conceived and designed to isolate and identify new thermo- and ethanol-tolerant AAB from Opuntia ficus-indica L. fruits. As a result, among 140 isolated bacterial strains, five selected strains (CR1, CR5, CR23, CZ2, and CZ15) exhibited important acetic acid production until 40°C. The use of 16S rDNA gene analysis was insufficient to identify selected bacteria. Indeed, except CR5 that presented 100% similarity to A. cerevisiae, the other strains presented similar homology rates simultaneously to the 16S rDNA sequences of A. cerevisiae and A. malorum. The reidentification by 16S-23S rDNA gene sequencing showed that CR1, CR23, and CZ15 were A. malorum, which were shown tolerance to the highest concentration of ethanol (12%) and produced elevated amount (40g/L) of acetic acid at 37°C. In summary, we showed the thermotolerance and ethanol tolerant character of new A. malorum strains, which can be used as a starter for vinegar production. Furthermore, during the molecular characterization of the isolated strains, we concluded that 16S-23S rDNA internal transcribed spacer sequence is of great importance for discriminating between AAB species as a complement to the identification by 16S rDNA sequencing.

Diversity of foliar endophytes in a dioecious wild grass and their interaction with the systemic Epichloë

Abstract This study compares the culturable fungal endophytic community inhabiting leaves of Epichloe-infected (E+) and Epichloe-free (E-) plants of Poa bonariensis. Plants were collected from a wild population (dioecious species) where E+ and E- plants co-exist. Fungal endophytes were isolated and then morphology and ribosomal DNA internal transcribed spacer sequences were used for the identification of isolates. In total 24 different taxa of non-Epichloe (NE) endophytes were identified. No significant differences were found in richness and diversity indexes of NE-endophytes between E+ and E- plants, or between pistillate (P) and staminate (S) plants. When we analyzed these effects based on the count of the most abundant genera, Alternaria and Stemphylium infection frequencies decreased in S plants, while in E+ plants, this tendency was observed for Alternaria. Our results suggest that Epichloe and the sex of the host plant may modulate some of the dominant foliar endophytic fungal taxa.

Increasing intensities of Anisakis simplex third-stage larvae (L3) in Atlantic salmon of coastal waters of Scotland

BackgroundRed Vent Syndrome (RVS), a haemorrhagic inflammation of the vent region in Atlantic salmon, is associated with high abundance of Anisakis simplex (s.s.) third-stage larvae (L3) in the vent region. Despite evidence suggesting that increasing A. simplex (s.s.) intensity is a causative factor in RVS aetiology, the definitive cause remains unclear.MethodsA total of 117 Atlantic salmon were sampled from commercial fisheries on the East, West, and North coasts of Scotland and examined for ascaridoid parasites. Genetic identification of a subsample of Anisakis larvae was performed using the internal transcribed spacer (ITS) region of ribosomal DNA. To assess the extent of differentiation of feeding grounds and dietary composition, stable isotope analysis of carbon and nitrogen was carried out on Atlantic salmon muscle tissue.ResultsIn the present study, the obtained ITS rDNA sequences matched A. simplex (s.s.) sequences deposited in GenBank at 99–100%. Not all isolated larvae (n = 30,406) were genetically identified. Therefore, the morphotype found in this study is referred to as A. simplex (sensu lato). Anisakis simplex (s.l.) was the most prevalent (100%) nematode with the highest mean intensity (259.9 ± 197.3), in comparison to Hysterothylacium aduncum (66.7%, 6.4 ± 10.2) and Pseudoterranova decipiens (s.l.) (14.5%, 1.4 ± 0.6). The mean intensity of A. simplex (s.l.) represents a four-fold increase compared to published data (63.6 ± 31.9) from salmon captured in Scotland in 2009. Significant positive correlations between A. simplex (s.l.) larvae intensities from the body and the vent suggest that they play a role in the emergence of RVS. The lack of a significant variation in stable isotope ratios of Atlantic salmon indicates that diet or feeding ground are not driving regional differences in A. simplex (s.l.) intensities.ConclusionsThis paper presents the most recent survey for ascaridoid parasites of wild Atlantic salmon from three coastal regions in Scotland. A significant rise in A. simplex (s.l.) intensity could potentially increase both natural mortality rates of Atlantic salmon and possible risks for salmon consumers due to the known zoonotic role of A. simplex (s.s.) and A. pegreffii within the A. simplex (s.l.) species complex.

Bioactivity assessment of endophytic fungi associated with Citrus macroptera Montr.: an endangered ethnomedicinal plant used in folk medicines in North-East India

Thirteen endophytic fungal isolates, inhabiting Citrus macroptera growing in Assam, India, were studied for their ability to produce bioactive metabolites. Ethyl acetate extracted secondary metabolites of endophytic Talaromyces assiutensis, exhibited the highest antimicrobial activity against Staphylococcus epidermidis followed by Acremonium falciformae which inhibited Sclerotinia sclerotiorum. Taxonomic identity of T. assiutensis was confirmed by internal transcribed spacer r-DNA sequence analysis. Quantitative estimation of phytochemicals of the ethyl acetate extracted secondary metabolites produced by T. assiutensis, revealed that flavonoid content was the highest followed by alkaloids, saponin and terpenoids. FT-IR spectrophotometry of the extract showed the presence of alcohol, alkane, acid anhydride, alkene, aromatic, alkyl halide and amine as functional groups. Nine major cmpounds, viz., 3-undecene-5-methyl-; 2,5-dihydroxy-3-methyl-2,5-cyclohexadiene-1,4-dione; 3-n-hexylthiolane, S,S-dioxide; Methyl 8-Methylnonanoate; (4H)4a,5,6,7,8,8a-Hexahydrobenzopyran-5-one-3-carboxamide,2; Cyclopentane undecanoic acid, methyl ester; Trans-2,4-Dimethylthiane, S,S-dioxide; 5-Hydroxy-4-hydroxymethyl-1-(1-hydroxy-1-isopropyl)-Cyclohex-3-ene and 3-Nonyn-1-ol were identified from the bioactive metabolites of T. assiutensis using GC–MS. The results indicate that the antimicrobial compounds produced by T. assiutensis may be used in pharmaceutical as well as agricultural industries.

Notes on Medicinal Polypore Species from the Genus Daedaleopsis (Agaricomycetes), Distributed in the Asian Part of Russia.

We present a study on the medicinal value, taxonomy, and ecology of the polypore mushrooms Daedaleopsis confragosa and D. tricolor isolated from the Asian part of Russia (the Urals, Siberia, and the Far East). The phylogenetic analysis of recombinant DNA internal transcribed spacer sequences data has shown that D. confragosa and D. tricolor do not differ taxonomically and should be considered as one species. However, because D. confragosa and D. tricolor differ significantly in their ecological characteristics, they may be considered as two morpho-ecological varieties: D. confragosa var. confragosa and D. confragosa var. tricolor (both nomen provisiorum).

Phylogenetic Study of Curvularia on Sorghum from Indonesia Based on ITS rDNA Sequence

Seven species of Curvularia were recorded from Indonesia, including species that pathogen to sorghum (Sorghum bicolor). The present study aims to determine four isolates of Curvularia isolated from sorghum in Indonesia using a combination of molecular phylogenetic analysis based on internal transcribed spacer (ITS) rDNA sequence and morphological examination. The results showed that the four isolates were genetically closed to C. lunata and C. dactyloctenicola. The morphological examination also insufficient to determine the identity of the four Curvularia isolates from sorghum until species level. Therefore, additional sequences from the partial fragments of the glyceraldehyde-3-phosphate dehydrogenase and the translation elongation factor 1-a genes are necessary to determine the identity of the Curvularia sequences isolated from sorghum in Indonesia until species level.