Subunit Ribosomal DNA Research Articles

Within-Site Variations in Soil Physicochemical Properties Explained the Spatiality and Cohabitation of Arbuscular Mycorrhizal Fungi in the Roots of Cryptomeria Japonica

Arbuscular mycorrhizal fungi (AMF) live in a community in the roots of host plants. Still, the patterns and factors that drive their spatiality and cohabitation remain uncovered, particularly that of trees in planted forests, which we aimed to clarify in Cryptomeria japonica, a major plantation tree in Japan. We analyzed 65 paired root and soil samples of Cryptomeria japonica trees collected from 11 microsite (MS) plots at two environmentally different forest sites in central Japan and measured soil pH, total phosphorus (TP), C, N, and the carbon-to-nitrogen ratio. Root AMF communities were recovered using Illumina’s next-generation amplicon sequencing targeting the small subunit of ribosomal DNA. We detected more than 500 AMF OTUs at each site but only three belonging to Dominikia, Rhizophagus, and Sclerocystis were dominant in the roots of C. japonica, detected each at an average relative abundance higher than 20%. Two showed negatively correlated spatial distributions and different associations with soil pH. Similarly, the physicochemical properties at MSs significantly determined the AMF assemblages in the roots of C. japonica. Dominikia, Rhizophagus, and Sclerocystis coexist in the roots of C. japonica where soil physicochemical properties, particularly pH, determine their spatial dynamic, turnovers, and cohabitation patterns. These findings highlight the importance of simultaneous colonization of plants by multiple AMF.

Three new species of Agaricus from Baiyun Mountain, Guangzhou, China

ABSTRACT Agaricus is a species-rich genus with more than 600 species around the world. In this work, three new species, Agaricus cacainus, A. baiyunensis, and A. praeclarefibrillosus are described from the specimens collected at Baiyun Mountain, Guangzhou, China, a subtropical area with a monsoon maritime climate, based on phylogenetic analyses and morphological examinations of internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), D1/D2 domains of the large subunit of ribosomal DNA (28S), and a part of translation elongation factor 1-alpha (TEF1). Agaricus cacainus in A. sect. Amoeni is characterized by a parabolic to applanate, slightly depressed pileus covered with chocolate brown, appressed, triangular squamules against white background, a white, furfuraceous stipe, an unchanging context when cut, a fragile and evanescent annulus, usually 4- or 2-spored basidia, and mostly pyriform cheilocystidia. Agaricus baiyunensis in A. sect. Minores has a pileus with a slightly truncate top covered with light brown, downy-wooly fibrillose scales and a light yellowish stipe with membranous annulus. Agaricus praeclarefibrillosus in A. sect. Brunneopicti is characterized by a pileus surface with brownish, triangular, recurved scales and longitudinally splitting lines toward margin, a cottony stipe with white, tiny, recurved fibrils, a single annulus, and variously shaped cheilocystidia, with sparsely ornamented basidiospores. The detailed comparison of their morphological characteristics with closely related species is provided.

Linking metacercariae and adults of Microphallus basodactylophallus (Digenea: Microphallidae), based on larval stages from ctenophores and adult parasites from aquatic birds found in Mexico

Members of the genus Microphallus Ward, 1901, are endoparasites mainly of birds and mammals distributed worldwide. Unencysted metacercariae of Microphallus sp., were collected from the mesoglea of ctenophores of the genus Pleurobrachia Fleming; adult digeneans were recovered from the intestines of Eudocimus albus Linnaeus (Threskiornithidae) and Buteogallus urubitinga Gmelin (Accipitridae), in four locations from southeastern Mexico. Adult specimens were identified as M. basodactylophallus (Bridgman, 1969) based on the following features: body pyriform entirely covered by minute spines, prepharynx short, oesophagus very long, caeca short and widely divergent, testes slightly symmetrical and excretory vesicle short and V-shaped. Sequences from D1–D3 domain of the large subunit of ribosomal DNA (LSU) were generated, aligned, and compared with those of congeneric species available in GenBank. Phylogenetic analyses indicated that the metacercariae and adults formed a clade together with an isolate identified as M. basodactylophallus from Florida, USA (GenBank: AY220628). The intraspecific genetic divergence among isolates was low ranged from 0.0% to 0.6%, allowing the link between the two stages of the life cycle. We observed phenotypic plasticity in the morphological traits of M. basodactylophallus adults in definitive hosts (mammals and birds) throughout the distribution, which ranged from the USA to southeastern Mexico. Finally, the unencysted metacercariae identified as M. basodactylophallus represent the first report of a microphallid in ctenophores.

Myxozoan Ceratomyxids Infecting the Gallbladder of Amazonian Ornamental Cichlid Fish: Description of Ellipsomyxa santarenensis n. sp. and Report of Ceratomyxa amazonensis in a New Host

Although most Myxozoa species of the genera Ceratomyxa and Ellipsomyxa have been described in marine hosts worldwide, an increasing diversity has been reported infecting South American freshwater fish, mainly in Amazonian waters. The present study deals with two species of myxozoan ceratomyxids parasitizing the gallbladder of Amazonian ornamental cichlids fish: Ceratomyxa amazonensis is identified from a new host—Geophagus altifrons; while Ellipsomyxa santarenensis n. sp. is described infecting Satanoperca jurupari. Morphological (light microscopy and transmission electron microscopy), molecular (small ribosomal subunit DNA—SSU-rDNA sequencing) and phylogenetic analyses were used to characterize both species. Ceratomyxa amazonensis showed a prevalence of 64.2%, with plasmodia showing a vermiform shape and motility. For E. santarenensis n. sp., the prevalence was 33.3%. Ultrastructural analysis revealed that the vermiform C. amazonensis plasmodia were composed of an outer cytoplasmic region and a large vacuole occupying the inner area. In E. santarenensis n. sp., cytoplasmic expansions were observed in pseudoplasmodia originating pseudopodia. SSU rDNA sequencing-based genetic distance analysis revealed a very small difference between C. amazonensis, parasite of G. altifrons, and C. amazonensis, parasite of S. discus—host of the original description, thus showing that they are the same species occurring in a new host. For Ellipsomyxa santarenensis n. sp., molecular data revealed a difference of 1.6% for Ellipsomyxa amazonensis and Ellipsomyxa paraensis. The phylogenetic analysis revealed the grouping of E. santarenensis n. sp. together with the other freshwater Ellipsomyxa species of the Amazonian region, and associated with the morphological data, it was possible to identify it as a new taxon within the genus Ellipsomyxa.

A molecular and ecological study of Macracanthorhynchus ingens (von Linstow, 1879) (Acanthocephala: Archiacanthocephala), in its paratenic and definitive hosts in southeastern Mexico and the Eastern USA.

The acanthocephalan Macracanthorhynchus ingens (von Linstow 1879) (Acanthocephala: Archiacanthocephala) is a parasite that infects the gut of carnivores (racoons, coyotes, wolves, foxes, badgers, skunks, opossum, mink and bears) as an adult and the body cavity of lizards, snakes, and frogs as a cystacanth in the Americas. In this study, adults and cystacanths of M. ingens from southeastern Mexico and southern Florida, USA, were identified morphologically by having a cylindrical proboscis armed with 6 rows of hooks each with 6 hooks. Hologenophores were used to sequence the small (SSU) and large (LSU) subunits of ribosomal DNA and cytochrome c oxidase subunit 1 (cox 1) from mitochondrial DNA.Phylogenetic analysis of the new SSU and LSU sequences of M. ingens placed them in a clade with other sequences available in GenBank identified as M. ingens. The cox 1 tree showed that the nine new sequences and six previously published sequences of M. ingens from the USA form a clade with other sequences previously identified as M. ingens from GenBank. The intraspecific genetic divergence among isolates from the Americas ranged from 0 to 2%, and in combination with the phylogenetic trees confirmed that the isolates belonged to the same species. The cox 1 haplotype network inferred with 15 sequences revealed 10 haplotypes separated from each other by a few substitutions. Rio Grande Leopard Frogs and Vaillant´s Frogs harbored cystacanths with low prevalence, 28% and 37% respectively, in Mexico. Brown Basilisks, an invasive lizard in Florida, USA, had high values of prevalence, 92% and 93% in males and females, respectively. Females harbored more cystacanths than males (0-39 vs 0-21) for unknown reasons that may, however, be related to ecological differences.

Mitochondrial COI and Cytb gene as valid molecular identification marker of sandfly species (Diptera: Psychodidae) in China

The accurate identification of sandfly species is crucial because some species transmit medically significant diseases, including leishmaniasis, bartonellosis and sandfly fever. However, due to the high similarity of the external morphology in sandfly species, identification can only be performed using internal morphological characteristics after dissection, which is time consuming and requires highly experienced staff. Thus, the introduction of suitable molecular markers may solve these identification problems. This study screened suitable DNA barcodes to identify common sandfly species in China. The phlebotomine sandflies were collected from Sichuan, Henan and Hainan Provinces from 2014 to 2016. The species were identified by the morphological characteristics of the pharyngeal armature and spermatheca. The genomic DNA of sandfly was extracted individually, and mitochondrial DNA (mtDNA) cytochrome C oxidase subunit I (COI) and cytochrome B (Cytb) as well as the 18S subunit of ribosomal DNA (rDNA) were amplified using polymerase chain reaction (PCR). Additionally, intraspecific and interspecific differences (p-distance) were calculated to evaluate the feasibility of the three gene fragments as a DNA barcode. The phylogeny trees of all sandfly species in this study were constructed using neighbor joining (NJ) method. Six species were identified by the morphological features, belonging to Phlebotomus and Sergentomyia, as Ph. chinensis s. l., Ph. stantoni, Se. bailyi, Se. iyengari, Se. squamirostris, and Se. squamipleuris. Analysis based on three gene fragments revealed some degree of intraspecific polymorphism among these sandfly species in China. The largest intraspecific variation occurred in Ph. chinensis s. l. (mtDNA COI, p-distance = 0.042; mtDNA Cytb, p-distance = 0.071), but the 18S rDNA fragment showed a small variation (p-distance = 0.005). The ranges of interspecific p-distances for mtDNA COI and mtDNA Cytb were 0.138 - 0.231 and 0.128 - 0.274, respectively. However, the interspecific p-distances of 18S rDNA are relatively low ranging from 0.003 to 0.055. Both mitochondrial COI and Cytb gene fragments are valid molecular identification markers in theses sandfly species. The topological structure of phylogeny trees based on mtDNA COI, mtDNA Cytb and 18S rDNA genes were all consistent with morphological classification. And we also found there were significant intraspecies differences within Ph. chinensis s. l. (0.006-0.071) and Se. bailyi (0.002-0.032) based on mtDNA Cytb gene fragment. Sequence alignment data suggested that Ph. chinensis s. l. from Sichuan should be Ph. sichuanensis, and the sandfly specimen collected from Henan was Ph. chinensis s. s.. There could be cryptic species in Se. bailyi from China.

P142 Lodderomyces elongisporus: A n emerging cause of fung emia

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMBackground Lodderomyces elongisporus, earlier considered as a sexual state of Candida parapsilosis, was described as a distinct species based on ribosomal RNA gene sequencing. Few cases of human infections by this yeast have been described from Mexico, China, Malaysia, Kuwait, Australia, and the USA. We describe here eight cases of fungemia by L. elongisporus from a tertiary care hospital in North India.MethodsClinical characteristics and risk factors associated with L. elongisporus fungaemia were evaluated. Yeast isolated from blood culture (BD BACTEC™ 9240, New Jersey, USA) was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS, Bruker Daltonik GmbH, Bremen, Germany) and sequencing of D1/D2 region of a large subunit of ribosomal DNA. We performed antifungal susceptibility testing for amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, caspofungin, anidulafungin, and micafungin by the microbroth dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI).ResultsWe report eight cases of fungemia caused by L. elongisporus at our tertiary care center. Of these, three were infants (males) and five were adults (3 males and 2 females). The mean age of adults was 43.4 years. Among the pediatric cases, underlying diseases included congenital heart disease/atrophic kidney (neonate), tracheoesophageal fistula (4 months), and late-onset neonatal sepsis (LONS). Among the adults, underlying illnesses included acute kidney injury (n = 2), superior mesenteric artery thrombosis with bowel gangrene (n = 1), diabetes (n = 1), and central nervous system (CNS) lymphoma (n = 1). The iatrogenic factors included the history of surgery (n = 3), admission to ICU (n = 3), presence of urinary catheter (n = 4), and presence of central venous catheter (n = 1). Seven patients were on broad-spectrum antibiotics. The mean stay in the hospital was 20.38 days. Three of the patients were managed with fluconazole. Six patients improved while one left against medical advice (LAMA) and one expired. The range of minimum inhibitory concentration (MIC) of all the isolates against antifungals was as follows: amphotericin B (0.03-0.25 μg/ml), fluconazole (0.125 μg/ml), voriconazole (0.03 μg/ml), itraconazole (0.03-0.06 μg/ml), posaconazole (0.03-0.25 μg/ml), caspofungin (0.03-0.06 μg/ml), micafungin (0.03-0.06 μg/ml), and anidulafungin (0.03 μg/ml).Conclusion Lodderomyces elongisporus is an emerging pathogenic yeast causing fungemia in patients with comorbidities and undergoing surgery or invasive interventions. Though no antifungal breakpoints exist for this yeast, all the isolates exhibited low MICs to all the tested antifungals.

Potential of Turkish Beauveria bassiana isolates for the management of the polyphagous planthopper, Orosanga japonica Melichar 1898 (Hemiptera: Ricaniidae)

BackgroundEntomopathogenic fungi are a potential biological control agent for many pests. In this study, 14 native Beauveria bassiana isolates were molecularly identified and assessed for their virulence and mortality against adults of the polyphagous planthopper Orosanga japonica, Melichar (Hemiptera: Ricaniidae) a polyphagous sap-feeding insect, under laboratory conditions.ResultsIsolates obtained from naturally infected adults O. japonica were molecularly identified as B. bassiana by sequencing the internal transcribed spacer and 26S large subunit of ribosomal DNA. In the bioassay, the radial growth and sporulation of isolates significantly differed (P < 0.05). Concentration–time bioassays demonstrated that all isolates had a lethal effect on adult O. japonica at the concentration of 1 × 108 conidia ml–1. The lethal times LT50 and LT90 values for each B. bassiana isolate, which indicate the time required to kill O. japonica, ranged between 2.44–3.19 and 3.78–5.01 days, respectively, at 20 °C, while their values were 1.74–2.76 and 2.76–4.10 days, respectively, at 25 °C depending on the native isolates. The concentration–mortality response showed a statistically significant difference between some isolates B. bassiana tested against adult O. japonica individuals (P < 0.05). With an LC50 value of 2.29 × 106 conidia ml–1, the lowest effective EPF isolate for O. japonica was KA-78-14. The isolates caused 100% adult mortality in O. japonica within five days, while the mortality in the control group was less than 10%.ConclusionsThese findings suggest that some B. bassiana isolates were more virulent on O. japonica and may play an important role in the biocontrol of O. japonica in Turkey.

First Report of Neonectria punicea Causing Preharvest Fruit Rot of Apple in Sichuan, China.

Apple (Malus domestica Borkh.) is an economic backbone driving poverty alleviation and rural revitalization in remote rural areas of China. Monitoring of pathogens associated with apple is particularly important for the apple industry. In 2019, survey for preharvest fruit diseases of apple was conducted in Yanyuan County, which lies on the southern edge of the Qinghai-Tibet Plateau, Sichuan, China. In one commercial orchard (27°26'49.8"N, 101°39'38.9"E), an uncharacterized fruit rot disease was observed on 'Red Fuji Nagafu No. 2' apple with prevalence of approximately 3%. Symptoms on the fruits occurred as light brown spots about 3 millimeters in diameter. With the progression of the disease, the originally infected sites turned deep brown, lesions expanded and decayed tissues became spongy. Ultimately, multiple lesions coalesced together, severely infected fruits became sunken, soft, wrinkled, and decayed completely. Twenty samples with typical symptoms were collected. Symptomatic apple tissues from the lesion margin were cut into 5 × 5 mm pieces, washed two times with 70% ethanol, disinfected with 0.5% NaClO for 5 min, rinsed twice with sterile double-distilled water, and plated on potato dextrose agar (PDA) medium at 20 °C in darkness for 5 days. Forty-five colonies were isolated from symptomatic tissues on PDA after 3 days and they shared similar morphology. Five single-spore isolates were obtained and a representative isolate designated ABT12-1 was characterized further (CCTCC accession no. M2021680) (Chaverri et al. 2011). The fungal colonies produced radial, abundant cottony aerial mycelium, white-saffron to saffron-ochraceous in color, and the reverse colony was ochraceous to sienna with yellow pigmentation in the agar. Conidiophores were observed developing laterally from aerial hyphae, irregularly branched or in fascicles, 1-4 branched, 21.5 to 87.1 μm long by 3.5 to 7.8 μm wide (n=20). Phialides were generally long and cylindrical or somewhat flask-shaped. Microconidia were generally ellipsoidal, with 0-1 septations and measured 3.2 to 16.6 × 2.5 to 7.2 μm (n=20). Macroconidia were ellipsoidal, oblong to long cylindrical, hyaline, smooth, slightly curved and round at both ends, with 3-7 septations, and averaged 45 × 4 μm (n=20). The fungus was initially identified as a member of the genus Neonectria based on the morphological observations (Lombard et al. 2015). Molecular identification was performed to determine fungal species by sequencing partial actin (Act), internal transcribed spacer (ITS), large subunit of ribosomal DNA (LSU), RNA polymerase II subunit one (Rpb1), translation elongation factor 1-α (Tef1), and the beta-tubulin (Tub) genes, which were amplified with primers Tact1/Tact2, ITS5/ITS4, LR5/LROR, crpb1a/rpb1c, tef1-728/tef1-986 and Btub-T1/Btub-T2, respectively (Carbone and Kohn 1999; Castlebury et al. 2006; O'Donnell and Cigelnik 1997; Schoch et al. 2012; White et al. 1990). The resulting sequences were deposited in GenBank (accession no. MW538899 for Act; MW534396 for ITS; MW534401 for LSU; MW538900 for Rpb1; MW538901 for Tef1; and MW538902 for Tub). Multilocus phylogenetic analysis with neighbor-joining methods based on the above 6 gene sequences showed that the fungal isolate clustered with Neonectria punicea (J.C. Schmidt) Castl. & Rossman. To test the pathogenicity, ten freshly picked 'Red Fuji' apple fruits were wound-inoculated with 2 µl of conidial suspension (1 × 105 conidia/ml) of the isolate ABT12-1; sterile water was used as control. The inoculated samples were placed in sterilized plastic boxes to maintain relative humidity and incubated at 20 °C for seven days. Pathogenicity experiments were conducted four times. After three days, the wound sites inoculated with conidial suspension showed visible diseased spots. As lesions progressively dilated, symptoms similar to those of the decayed fruit in the field were observed, whereas the control fruits remained symptomless. The pathogen was reisolated from symptomatic lesions, thus fulfilling Koch's postulates. Neonectria punicea causes stem and bark canker diseases on a variety of trees (Hirooka et al. 2013; Karadžić et al. 2020; Salgado-Salazar and Crouch 2019). To our knowledge, N. punicea has never been reported causing apple fruit rot in China.

Phylogenetic relationship of coffee leaf rust in the central jungle of Peru

Coffee leaf rust is the main disease that causes significant losses in Coffea arabica. In Peru, this disease caused epidemics between 2008 and 2013 with production losses of 35 %. The objective was to identify H. vastatrix using a morphological and molecular approach based on a phylogenetic species concept. Coffee leaf samples with symptoms of chlorotic lesions with the presence of yellow uredospores at different severity stages of different cultivars were collected from 11 locations in the departments of Pasco and Junin during 2017-2018. DNA was purified as proposed by Cristancho and coworkers. The major subunit of ribosomal DNA was amplified with universal primers LR0R and LR5, and sequenced by Macrogen and deposited in GenBank. Sequences from the genera Achrotelium, Blastospora, Cystopsora, Hemileia, and Mikronegeria were included for phylogenetic analysis. The results showed that the rust was distributed in coffee growing regions of Pasco: Villa Rica (Catimor, Caturra, and Gran Colombia); Oxapampa (Yellow Caturra), and Junín: San Luis de Shuaro (Catimor), Chanchamayo (Catimor), San Ramón (Catimor), Vitoc (Caturra), Pichanaki (Caturra), Río Negro (Caturra), Pangoa (Yellow Caturra, Gran Colombia, Limani). It was also grouped into a single clade with isolated H. vastatrix from Mexico and Australia, suggesting that they come from a common ancestor. This is the first confirmed report using molecular barcoding of H. vastatrix in the central jungle of Peru.

Molecular Diversity of Arbuscular Mycorrhizal Fungi (AMF) Associated with Carissa edulis, an Endangered Plant Species along Lake Victoria Basin of Kenya

Carissa edulis is a tropical plant belonging to the family Apocynaceae. The species is widely used in the preparation of various herbal medicines. Earlier works in Kenya show that an aqueous extract from the roots of C. edulis has remarkable anti-herpes simplex virus. Due to its medicinal value, the species has been overexploited in its natural range and requires conservation interventions. Studies show that the species has beneficial relationships with arbuscular mycorrhizal fungi (AMF) that can enhance restoration of its population; however, no study has been undertaken to document the diversity of these AMF species. This study evaluated the genetic diversity of AMF associated with the roots of C. edulis within Lake Victoria basin ecosystem of Kenya. A cross-sectional, laboratory-based prospective study was carried out from roots of C. edulis collected from six sites within the ecosystem. Root samples were collected from 6 points (replicates) per site. AMF was assessed through morphological characterization and sequencing of small subunit of ribosomal DNA. Morphological identification identified four genera of AMF (Gigaspora, Acaulospora, Scutellospora, and Glomus) with no significant difference among the sites. Molecular analysis also revealed presence of four genera, but only two (Glomus and Acaulospora) were common for both the analyses with Glomus as the most predominant genera. In all the sites, there were large numbers of spores both in soil and in the roots confirming the association between C. edulis and AMF.

Hepatic microsporidiosis of mudskipper, Boleophthalmus dussumieri Valenciennes, 1837 (Perciformes: Gobiidae), due to Microgemma sp.

The present study reports a case of hepatic microsporidiosis caused by Microgemma sp. in brackishwater fish, Boleophthalmus dussumieri (Valenciennes, 1837) (n = 60), from thewest coast of India. An eight-month study from September 2017 to April 2018 revealed a prevalence of 11.7% for this parasite. The microsporidian showed tissue-specific infection and did not reveal any gross pathology in infected fish. Small whitish cysts containing microspores of size 0.3-0.5mm were observed in the liver of fish. The range of pyriform microsporidian spore size varied from 2.9-3.77×1.85-2.67µm. Scanning electron microscopy of the spores showed a distinct groove on the anterior end of the spore for polar tube extrusion. Polymerase chain reaction (PCR) amplification of the DNA extracted from the microsporidian-infected liver tissue using primers targeting small ribosomal subunit DNA (SSU rDNA) yielded ~ 1340bp amplicon and the genetic distance analysis showed a 0.2% variation with the reported M. tilanpasiri. Accordingly, in the phylogenetic tree, the present species of Microgemma clustered with M. tilanpasiri. Even though, the morphomeristic characters of the present Microgemma sp. was marginally different from the reported M. tilanpsasiri; the SSU rDNA showed considerably higher similarity with M. tilanpasiri. Thus, we report the species of Microgemma as Microgemma aff. tilanpasiri from a new host. This is the first report of a microsporidian from B. dussumieri and the first record of the genus Microgemma from India.

In Vitro Evaluation of Probiotic Properties of Indigenous Yeasts Isolated from Nigerian Fermented Food Products

Probiotics are living microbial food supplements which beneficially affect the host by improving the intestinal microbial balance. Before an organism can be designated as probiotic there are certain criteria that must be fulfilled. These include acid and bile tolerance, antimicrobial activity, ability to co-aggregate, hydrophobicity etc. One hundred and eighty one indigenous yeast isolates recovered from various fermented food products of Nigeria were characterized and grouped using phenotypic methods. Forty two selected yeast isolates were identified using molecular method which involved sequencing of D1 and D2 domain of the large subunit of ribosomal DNA. Then nine indigenous Saccharomyces cerevisiae were evaluated for their probiotic characteristics such as acid and bile tolerance, transit in simulated gastric and intestinal juices, autoaggregation and hydrophobicity. Saccharomyces cerevisiae SC10 was included as a positive control. The S. cerevisiae were able to grow in the presence of acidic medium with pH as low as 2 and 3. In the minimum inhibitory concentration test with 0-1% ox bile, all the S. cerevisiae tested were able to grow. The growth for 3% bile tolerance test ranged from 4.81 to 5.35 log cfu/ml. These isolates were able to survive in simulated gastro-intestinal transit. All the yeast isolates exhibited bile salt deconjugation activity against sodium glycodeoxycholate and were able to grow in the presence of all other bile salts investigated. Autoaggregation ability (an adhesive property) of the indigenous yeast isolates ranged from 89.80% for S. cerevisiae BK19 to 99.91% for S. cerevisiae OB03. The native yeast isolates also exhibited high percentage hydrophobicity, another adhesive property of probiotics. The values obtained ranged from 31.62 to 83.45% for isolates AG23A and OB 17. These observations indicate that the native yeast isolates from Nigerian fermented foods have the potential of being use as probiotics for making functional foods.

Cladophora rigida sp. nov., a new freshwater species within Cladophoraceae (Ulvophyceae, Chlorophyta) from China

ABSTRACT Cladophora is one of the most species-rich and taxonomically challenging taxa of green algae. Recently, some samples of freshwater filamentous algae were collected from Hubei, Yunnan and Guizhou Provinces, China. The specimens grew attached to solid substrates by basal rhizoids or holdfasts, and their thalli were bright to dark green, long and stiff. They were typically composed of uniseriate unbranched filaments of approximately equal length produced by intercalary and apical cell divisions. We studied the taxonomy of these algae based on morphological and molecular data. Microscopic observations showed that the filaments had some characteristics that differed from those of other species of Cladophora, such as being unbranched, with a large cell diameter (180–429 µm) and smaller length:diameter (L:D) ratio (0.5–3.3) than other species. Phylogenetic trees constructed on the basis of small (SSU) and large (LSU) subunits of ribosomal DNA (rDNA) and the internal transcribed spacer (ITS) sequences showed that all collected specimens belonged to one species. These specimens formed a new clade in the freshwater Cladophora lineage. On the basis of the morphological and phylogenetic analyses, we propose a new species, Cladophora rigida sp. nov.

First steps to understand the systematics of Echinorhynchidae Cobbold, 1876 (Acanthocephala), inferred through nuclear gene sequences.

Acanthocephalans of the order Echinorhynchida are one of the most diverse groups in their phylum, with approximately 470 species classified into 11 families that largely consist of parasites of freshwater, brackish and marine fishes and, sporadically, reptiles and amphibians distributed worldwide. Previous phylogenies inferred with molecular data have supported the paraphyly or polyphyly of some families, suggesting that most of them have been diagnosed based on unique combinations of characters, rather than shared derivative features. We expand the taxonomic sampling of several genera such as Acanthocephalus, Echinorhynchus and Pseudoacanthocephalus of Echinorhynchidae from diverse biogeographical zones in the Americas, Europe and Asia with the aim of testing the monophyly of the family by using two molecular markers. Sequences from small (SSU) and large (LSU) subunits of ribosomal DNA were obtained for six species representing the genera Acanthocephalus and Echinorhynchus from the Neotropical, Nearctic, Palearctic and Oriental regions. These sequences were aligned with other sequences available in the GenBank dataset from Echinorhynchidae. Phylogenetic trees inferred with the combined (SSU+LSU) and the individual data sets consistently placed the genera Acanthocephalus, Pseudoacanthocephalus and Echinorhynchus into three independent lineages. Two families, Paracanthocephalidae Golvan, 1960, and Pseudoacanthocephalidae Petrochenko, 1956, were resurrected to accommodate the genera Acanthocephalus and Pseudoacanthocephalus, respectively. The species of the genus Acanthocephalus from the Nearctic, Palearctic and Oriental biogeographic regions formed a clade that was well supported. However, Acanthocephalus amini from the Neotropical region was nested inside Arhythmacanthidae. Therefore, the genus Calakmulrhynchus was created to accommodate A. amini and resolve the paraphyly of Acanthocephalus. Finally, the diagnoses of the families Echinorhynchidae and Arhythmacanthidae were amended. The molecular phylogenies should be used as a taxonomic framework to find shared derived characters (synapomorphies) and build a more robust classification scheme that reflects the evolutionary history of the acanthocephalans.

Morphological and molecular characterization of Cytospora species involved in apple decline in Iran

In a recent survey on apple orchards showing stem canker, dieback and decline symptoms in West Azerbayjan province, Iran, several fungal isolates with typical characteristics of the genus Cytospora were obtained. Combination of morphological and cultural characteristics and phylogenetic analysis of internal transcribed spacer region of the nrDNA (ITS-rDNA) and parts of large subunit of ribosomal DNA (LSU), actin (ACT) and RNA polymerase II (RPB2) genes were used to accurate delimitation of fungal species. Four Cytospora species viz. C. chrysosperma, C. germanica, C. paratranslucens and C. salicina were identified. Pathogenicity tests were conducted on detached branches of ‘Golden delicious’ and ‘Red delicious’ apple cultivars. Isolates of C. salicina caused characteristic lesions on both ‘Golden delicious’ and ‘Red delicious’ apple cultivars, while isolates of C. chrysosperma, C. germanica and C. paratranslucens were only pathogenic on ‘Red delicious’ apple cultivar. Re-isolation and identification of the inoculated fungi confirmed Koch’s postulates. This study indicated the presence of different Cytospora species causing apple canker, dieback and decline disease in the studied area. Cytospora germanica and C. salicina are reported for the first time as causal agents of apple canker disease. Moreover, these species are reported as new records for the mycobiota of Iran.

Phylogenetic Assignment of the Fungicolous Hypoxylon invadens (Ascomycota, Xylariales) and Investigation of its Secondary Metabolites.

The ascomycete Hypoxylon invadens was described in 2014 as a fungicolous species growing on a member of its own genus, H. fragiforme, which is considered a rare lifestyle in the Hypoxylaceae. This renders H. invadens an interesting target in our efforts to find new bioactive secondary metabolites from members of the Xylariales. So far, only volatile organic compounds have been reported from H. invadens, but no investigation of non-volatile compounds had been conducted. Furthermore, a phylogenetic assignment following recent trends in fungal taxonomy via a multiple sequence alignment seemed practical. A culture of H. invadens was thus subjected to submerged cultivation to investigate the produced secondary metabolites, followed by isolation via preparative chromatography and subsequent structure elucidation by means of nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). This approach led to the identification of the known flaviolin (1) and 3,3-biflaviolin (2) as the main components, which had never been reported from the order Xylariales before. Assessment of their antimicrobial and cytotoxic effects via a panel of commonly used microorganisms and cell lines in our laboratory did not yield any effects of relevance. Concurrently, genomic DNA from the fungus was used to construct a multigene phylogeny using ribosomal sequence information from the internal transcribed spacer region (ITS), the 28S large subunit of ribosomal DNA (LSU), and proteinogenic nucleotide sequences from the second largest subunit of the DNA-directed RNA polymerase II (RPB2) and β-tubulin (TUB2) genes. A placement in a newly formed clade with H. trugodes was strongly supported in a maximum-likelihood (ML) phylogeny using sequences derived from well characterized strains, but the exact position of said clade remains unclear. Both, the chemical and the phylogenetic results suggest further inquiries into the lifestyle of this unique fungus to get a better understanding of both, its ecological role and function of its produced secondary metabolites hitherto unique to the Xylariales.

A new species of Gloeandromyces from Ecuador and Panama revealed by morphology and phylogenetic reconstruction, with a discussion of secondary barcodes in Laboulbeniomycetes taxonomy

ABSTRACT This paper describes and illustrates a new species of Laboulbeniales (Ascomycota, Laboulbeniomycetes) recovered from Mastoptera guimaraesi bat flies (Diptera, Streblidae) in Ecuador and Panama. Bat fly–associated Laboulbeniales are still unexplored in the Neotropics, with only four described species of Gloeandromyces and one species of Nycteromyces known. Morphological characteristics and phylogenetic analyses support placement of the new taxon in Gloeandromyces and its recognition as an undescribed species. Gloeandromyces hilleri sp. nov. is easily recognized by 2–3 longitudinal rows of undulations at its perithecial venter. Phylogenetic reconstructions of the large subunit (LSU) ribosomal DNA and the translation elongation factor 1α (TEF1) both resolve G. hilleri and G. nycteribiidarum as sister species. We discuss the utility of LSU and TEF1 as secondary barcodes in Laboulbeniomycetes taxonomy.

Characterization of Amylolytic Activity by a Marine-Derived Yeast Sporidiobolus pararoseus PH-Gra1

Marine yeasts have tremendous potential in industrial applications but have received less attention than terrestrial yeasts and marine filamentous fungi. In this study, we have screened marine yeasts for amylolytic activity and identified an amylase-producing strain PH-Gra1 isolated from sea algae. PH-Gra1 formed as a coral-red colony on yeast–peptone–dextrose (YPD) agar; the maximum radial growth was observed at 22 °C, pH 6.5 without addition of NaCl to the media. Based on the morphology and phylogenetic analyses derived from sequences of internal transcribed spacer (ITS) and a D1/D2 domain of large subunit of ribosomal DNA, PH-Gra1 was designated Sporidiobolus pararoseus. S. pararoseus is frequently isolated from marine environments and known to produce lipids, carotenoids, and several enzymes. However, its amylolytic activity, particularly the optimum conditions for enzyme activity and stability, has not been previously characterized in detail. The extracellular crude enzyme of PH-Gra1 displayed its maximum amylolytic activity at 55 °C, pH 6.5, and 0%–3.0% (w/v) NaCl under the tested conditions, and the activity increased with time over the 180-min incubation period. In addition, the crude enzyme hydrolyzed potato starch more actively than corn and wheat starch, and was stable at temperatures ranging from 15 °C to 45 °C for 2 h. This report provides a basis for additional studies of marine yeasts that will facilitate industrial applications.

Prevalence and genotypic identification of Cryptosporidium spp. and Enterocytozoon bieneusi in captive Asiatic black bears (Ursus thibetanus) in Heilongjiang and Fujian provinces of China

BackgroundCryptosporidium and Enterocytozoon bieneusi are two important pathogens with zoonotic potential that cause enteric infections in a wide range of hosts, including humans. Both are transmitted from animals to humans by direct contact or through contaminated equipment. Bears are frequently found in Chinese zoos as ornamental animals as well as farmed as commercial animals, and are therefore in close contact with zoo- or farm-keepers, but the prevalence and zoonotic potential of Cryptosporidium and E. bieneusi in bears is poorly understood. In this study, we aimed to provide data on the occurrence and genetic diversity of Cryptosporidium and E. bieneusi in Asiatic black bears from Heilongjiang and Fujian, China. From May 2015 to December 2017, 218 fresh fecal specimens were collected from captive Asiatic black bears in Heilongjiang (n = 36) and Fujian (n = 182), China. Cryptosporidium and E. bieneusi were examined by PCR amplification of the partial small subunit of ribosomal DNA (SSU rDNA) and the internal transcribed spacer (ITS) region of rDNA, respectively. C. andersoni-positive isolates were subtyped through PCR analysis of the four minisatellite/microsatellite (MS1, MS2, MS3 and MS16) loci.ResultsThe overall prevalence of Cryptosporidium and E. bieneusi were 2.4% (4/218) and 6.4% (14/218), respectively, with 2.8% (1/36) and 22.2% (8/36) in the Heilongjiang Province, and 1.6% (3/182) and 3.3% (6/182) in the Fujian Province. Sequence analysis confirmed the presence of Cryptosporidium species: C. andersoni (n = 3) and a genotype termed Cryptosporidium rat genotype IV (n = 1). All three identified C. andersoni belonged to the MLST subtype A4, A4, A4, A1. Two known E. bieneusi genotypes D (n = 4) and SC02 (n = 10) were identified, both of which belong to zoonotic Group 1.ConclusionsThis is the first report of C. andersoni and Cryptosporidium rat genotype IV in bears. The discovery of the zoonotic potential of E. bieneusi genotype D in bears highlights its significant zoonotic potential and potential threat to human health.