ITS Gene Sequences Research Articles

Молекулярно-генетическая идентификация грибов рода Fomitopsis из Иркутской области

Basidiomycetes of the genus Fomitopsis are an extensive group of more than 100 species that have ecosystem significance and high biotechnological potential. One of these species, Fomitopsis pinicola, is known as a widespread tinder fungus, a saprophyte that causes brown rot on deciduous and coniferous trees. The modern taxonomy of F. pinicola, economic significance, relevance of pharmacological research and the possibility of using them as medicines and food additives are discussed. F. pinicola has an important ecosystem function as it promotes the nutrient circulation in the forest. Recent studies show that this is a species complex and that morphological markers alone are not sufficient for precise identification of the species, which emphasises the importance of a molecular genetic approach to the taxonomy of this group. Although F. pinicola can be detrimental to forestry by causing rotting of wood, this basidiomycete has long been used by humans. For a long time, extracts of the conks (fruiting bodies) of F. pinicola, like many other tinder fungi, have been used by various cultures in traditional medicine, but modern pharmacological studies of the health-saving properties of the substances produced by F. pinicola are still insufficient. However, the relevance of such studies is very high, since ergosterol, fomitopic acids, and fomitosides produced by fungi demonstrate a proapoptotic effect and affect the metabolic pathway of prostaglandins. Promisingly, some compounds isolated from other Fomitopsis species have anticancer properties. Despite this, in fungal taxonomy, the correct species identification of biotechnological strains remains a problem. Due to intraspecific morphological variation, modern taxonomy emphasizes genetic markers. Here the data of molecular-genetic and phylogenetic identification of two biotechnological strains of basidiomycetes collected from the affected larch stands of the Irkutsk region are presented. Genetic identification of strains of Fomitopsis spp. was carried out using the sequence of variable regions of the ITS1 ribosomal operon. For phylogenetic analysis, ITS1 gene sequences of 25 species of basidiomycetes evolutionarily closest to Fomitopsis spp. were used. The studied strains were identified as the F. pinicola species with an accuracy of 97.98% and 99.21%, respectively. Further study of the biotechnological properties of the two identified strains of F. pinicola seems to be very relevant.

First Report of Fusarium Stem and Root Rot of Gerbera jamesonii Caused by Fusarium incarnatum in China.

Gerbera (Gerbera jamesonii Bolus) is an important cut flower grown globally. In 2020, gerbera plants (Redaicaoyuan, Baimawangzi, and Hongditan cultivars) with roots, crowns, and stems rot were found in a greenhouse in Nanping, Fujian, China. Approximately 30% of the 60,000 plants showed symptoms. Diseased plants were stunted with chlorotic leaves. The leaves and flower heads were wilted and withered. Brown discoloration with red to black streaks occurred in the vascular system of the crown and stem. The stem pieces (3×3 mm) showing the symptom were surface-disinfected with 1% NaClO for 1 min and washed three times with sterilized water. The stem pieces were then dried and placed on potato dextrose agar (PDA) at 25℃ inside a dark chamber. Ten single-spored isolates were identified asFusarium incarnatumbased on morphological features. White to light brown mycelia were observed among the isolates on PDA medium. Falculate, multicelluar, straight to slightly curved macroconidia produced in monophialide sporodochia without distinctive foot shaped basal cell; and chlamydospores produced in some isolates (Leslie and Summerell). The size of macroconidia was 36.4 ± 5.20 × 4.6 ± 1.3 μm (n = 100) with 3 to 5 septates. Microconidia were mostly 0 to 1 septate measured 14.6 ± 1.9 × 2.6 ± 0.5 μm (n=100). Based on the morphological observation, isolates were further identified by molecular method. The ITS1/4 region combined with partial gene fragments of translation elongation factor (EF-1α, primer EF1/EF2, Geiser et al.) and calmodulin (CAM, primer CL1/CL2A, O'Donnell.) from the isolates were amplified and sequenced. All of the three tested isolates showed identical gene sequences. Sequences amplified from one represented isolate FIN-1 were submitted to Genbank. BLAST searches revealed that ITS1/4 (MW527088), EF-1α (MW556488), and CAM (MW556487) had 99.22%, 99.53%, 99.42% identity compared to F. incarnatum (MN480497, MN233577, and LN901596, respectively) in GenBank. FUSARIUM-ID (Geiser et al. 2004) analysis also showed 99 to 100% similarity with sequences of the F. incarnatum-equiseti species complex (FIESC) (FD_01636 for CAM, FD_01643 for EF-1α). The phylogenetic analysis was conducted using neighbor-joining algorithm based on the ITS, EF-1α, and CAM gene sequences. The isolate was clustered with F. incarnatum clade. Then, the pathogenicity of the fungus was confirmed by performing Koch's postulates. Pure single-spored cultures were grown on carboxymethyl-cellulose (CMC) medium for sporulation. G. jamesonii plants used for pathogenicity tests were grown on sterilized potting soil in a plastic container to the ten-leaf stage prior to inoculation. Spores harvested from the CMC medium were adjusted to a concentration of 1×105 conidial/ml. Twelve healthy rooted gerbera seedlings were inoculated by drenching 10 ml of the conidial suspension onto roots. Twelve gerbera seedlings treated with 10 ml sterile water served as control treatments. Plants were grown in the glasshouse at temperatures of 23°C, relative humidity >70%, and 16 h light per day. After 10 days, blackening stems and withered leaf edges began to appear on inoculated seedlings, whereas control seedlings remained healthy. F. incarnatum was consistently re-isolated from the symptomatic stems, whereas no isolates were obtained from the control seedlings. The assay was conducted twice. To the best of our knowledge, this is the first report of F. incarnatum causing stem and root rot on G. jamesonii.

Identification of Fusarium solani as a causal agent of black spot disease (BSD) of Pacific white shrimp, Penaeus vannamei

In Nov 2020, mass mortality of cultured Pacific white shrimp (Penaeus vannamei) occurred due to black spot disease (BSD) in some shrimp farms in China. To investigate the disease cause, a potential pathogenic fungus was isolated from melanized lesions of P. vannamei suffering from the BSD syndrome. The fungal isolate was then identified as Fusarium solani based on TEF1α and ITS-gene sequences. The isolate of fungus was used in infection studies. This study used Koch postulates confirmed that the studied agent was causing BSD symptoms in the white leg shrimp, which were significantly symptoms relieved after molting. Results showed that an identical fungus was re-isolated from black lesions after the artificial infection period. Virulence test revealed that F. solani could cause 88.66% mortality within 30 dpi at a dose of 1.02 × 106 CFU/mL, and the LC50 of F. solani at 30 dpi is 3.37 × 104 CFU/mL. The accumulative mortalities also displayed concentration dependence. The histopathological characterization displayed that responses of P. vannamei against F. solani are haemocytic infiltration, encapsulation, melanization, etc. in the black spot lesions. Furthermore, severe infection of F. solani in P. vannamei causes molting failure, and further contributes to the mortality of the diseased individuals. The findings of our study would be useful to provide a theoretical basis for early prevention and control of BSD.

Heterodera amaranthusiae n. sp. (Nematoda: Heteroderidae), a new cyst nematode parasitising Amaranthus retroflexus L. in China

Summary A new species of cyst-forming nematode, Heterodera amaranthusiae n. sp., is described and illustrated from the weed, Amaranthus retroflexus, in a potato field in Yunnan Province, China. It is characterised by having canary to russet-brown and asymmetric lemon-shaped cyst, distinct neck, bifenestrate vulval cone, relatively short vulval slit of 29 (28-32) μm, bullae absent and underbridge absent or weak if present. Second-stage juveniles are characterised by a well-developed stylet of 23 (22-25) μm with robust shaft and basal knobs concave anteriorly, tail conoid, 51 (48-58) μm long and hyaline region comprising 48 (41-53)% of its length. Morphologically and morphometrically it most resembles H. vallicola in the Humuli group. The ITS, 28S and COI gene sequences of H. amaranthusiae n. sp. clearly differentiate it from other Heterodera species. For diagnostic purposes, restriction enzyme analysis of the ITS region and three restriction enzymes, AluI, BsuRI (HaeIII) and CfoI (HhaI), were selected, clearly distinguishing H. amaranthusiae n. sp. from representative species in the Humuli group. Phylogenetic relationships with other species of the genus, inferred from two ribosomal regions and the cytochrome oxidase c subunit 1 region, based on Bayesian analysis, consistently showed that H. amaranthusiae n. sp. clustered with high support with other Humuli group species but with separate species status.

Leaf spot disease of Orychophragmus violaceus caused by Alternaria tenuissima in China.

Leaf spot disease of Orychophragmus violaceus caused by Alternaria tenuissima in China.

Intestinal microbiota changes induced by TNF-inhibitors in IBD-related spondyloarthritis

BackgroundThe close relationship between joints and gut inflammation has long been known and several data suggest that dysbiosis could link spondyloarthritis (SpA) to inflammatory bowel diseases (IBD). The introduction of...

Cystic Fibrosis Clinical Isolates of Aspergillus fumigatus Induce Similar Muco-inflammatory Responses in Primary Airway Epithelial Cells.

Aspergillus is increasingly associated with lung inflammation and mucus plugging in early cystic fibrosis (CF) disease during which conidia burden is low and strains appear to be highly diverse. It is unknown whether clinical Aspergillus strains vary in their capacity to induce epithelial inflammation and mucus production. We tested the hypothesis that individual colonising strains of Aspergillus fumigatus would induce different responses. Ten paediatric CF Aspergillus isolates were compared along with two systemically invasive clinical isolates and an ATCC reference strain. Isolates were first characterised by ITS gene sequencing and screened for antifungal susceptibility. Three clusters (A−C) of Aspergillus isolates were identified by ITS. Antifungal susceptibility was variable, particularly for itraconazole. Submerged CF and non-CF monolayers as well as differentiated primary airway epithelial cell cultures were incubated with conidia for 24 h to allow germination. None of the clinical isolates were found to significantly differ from one another in either IL-6 or IL-8 release or gene expression of secretory mucins. Clinical Aspergillus isolates appear to be largely homogenous in their mucostimulatory and immunostimulatory capacities and, therefore, only the antifungal resistance characteristics are likely to be clinically important.

Identification of a new Talaromyces strain DYM25 isolated from the Yap Trench as a biocontrol agent against Fusarium wilt of cucumber

Fusarium equiseti is a pathogenic fungus of plant root rot, and there are few studies on the biocontrol strains of plant wilt caused by F. equiseti. Hence, we conducted a screening and antimicrobial characterization study of marine-origin biocontrol fungi from water samples of the Yap Trench. A new Talaromyces strain DYM25 was screened from water samples of the Yap Trench in the western Pacific Ocean, and its potential as a biocontrol agent against Fusarium wilt of cucumber was studied. 18S rRNA and ITS gene sequencing verified that strain DYM25 belongs to the genus Talaromyces. The growth of F. equiseti was inhibited by strain DYM25 through the antibiosis effect. A preliminary test was first conducted to examine the bioactive stability of filtered DYM25 broth against F. equiseti under various conditions, including high temperature, UV light, alkaline environment, and the presence of metal ions, which indicated its potential as a bio-control agent. The results of the pot experiment showed that F. equiseti caused cucumber wilt, which could be mitigated using the fermentation broth of strain DYM25 (52.9 %). On the other hand, the alkaloid chromogenic reaction showed that the alkaloid salts present in the crude n-butanol extracts were most likely the major components that might have an antimicrobial effect. Therefore, Talaromyces sp. DYM25 represents a new species that can be used as a novel biocontrol agent against cucumber wilt.

Isolation and characterization of endophytic fungi having plant growth promotion traits that biosynthesizes bacosides and withanolides under in vitro conditions.

Endophytes are regarded with immense potentials in terms of plant growth promoting (PGP) elicitors and mimicking secondary metabolites of medicinal importance. Here in the present study, we explored Bacopa monnieri plants to isolate, identify fungal endophytes with PGP elicitation potentials, and investigate secretion of secondary metabolites such as bacoside and withanolide content under in vitro conditions. Three fungal endophytes isolated (out of 40 saponin producing isolates) from leaves of B. monnieri were examined for in vitro biosynthesis of bacosides. On morphological, biochemical, and molecular identification (ITS gene sequencing), the isolated strains SUBL33, SUBL51, and SUBL206 were identified as Nigrospora oryzae (MH071153), Alternaria alternata (MH071155), and Aspergillus terreus (MH071154) respectively. Among these strains, SUBL33 produced highest quantity of Bacoside A3 (4093 μg mL−1), Jujubogenin isomer of Bacopasaponin C (65,339 μg mL−1), and Bacopasaponin C (1325 μg mL−1) while Bacopaside II (13,030 μg mL−1) was produced by SUBL51 maximally. Moreover, these aforementioned strains also produced detectable concentration of withanolides—Withaferrin A, Withanolide A (480 μg mL−1), and Withanolide B (1024 μg mL−1) respectively. However, Withanolide A was not detected in the secondary metabolites of strain SUBL51. To best of our knowledge, the present study is first reports of Nigrospora oryzae as an endophyte in B. monnieri with potentials of biosynthesis of economically important phytomolecules under in vitro conditions.Supplementary InformationThe online version contains supplementary material available at 10.1007/s42770-021-00586-0.

Soil fungi are more sensitive than bacteria to short-term plant interactions of Picea asperata and Abies faxoniana

Despite increasing knowledge of plant interactions on the aboveground parts, little is known about the belowground mechanisms underlying such interactions. Here, we selected Picea asperata and Abies faxoniana, two dominant species of subalpine forests, to investigate the effects of planting patterns (isolation and intra- and interspecific interactions) on soil microbial communities. High-throughput 16S rRNA and ITS gene sequencing were performed to explore the bacterial and fungal community structures of the soils. The fungal community changed more under interactions of two species than isolation. Intra- and interspecific interactions had different effects on the fungal community. However, neither intra- nor interspecific interactions had effects on bacterial community composition. Compared with bacteria, fungi were more strongly affected by soil properties, such as the nitrate nitrogen concentration, total nitrogen concentration, pH, and β-1,4-N-acetyl-glucosaminidase activity. These results indicated that fungi were more susceptible than bacteria to plant interactions. Fungal community composition and structure, especially those of ectomycorrhizal fungi, were changed by intra- and interspecific interactions. In inter- and intraspecific interactions with P. asperata, the relative abundance of the ectomycorrhizal fungal Inocybe and Trichophaea increased, which may be one of important factors directly or indirectly affecting belowground interactions.

Fungal Associates of Soft Scale Insects (Coccomorpha: Coccidae).

Ophiocordyceps fungi are commonly known as virulent, specialized entomopathogens; however, recent studies indicate that fungi belonging to the Ophiocordycypitaceae family may also reside in symbiotic interaction with their host insect. In this paper, we demonstrate that Ophiocordyceps fungi may be obligatory symbionts of sap-sucking hemipterans. We investigated the symbiotic systems of eight Polish species of scale insects of Coccidae family: Parthenolecanium corni, Parthenolecanium fletcheri, Parthenolecanium pomeranicum, Psilococcus ruber, Sphaerolecanium prunasti, Eriopeltis festucae, Lecanopsis formicarum and Eulecanium tiliae. Our histological, ultrastructural and molecular analyses showed that all these species host fungal symbionts in the fat body cells. Analyses of ITS2 and Beta-tubulin gene sequences, as well as fluorescence in situ hybridization, confirmed that they should all be classified to the genus Ophiocordyceps. The essential role of the fungal symbionts observed in the biology of the soft scale insects examined was confirmed by their transovarial transmission between generations. In this paper, the consecutive stages of fungal symbiont transmission were analyzed under TEM for the first time.

Isolation and Characterization of Nickel-Tolerant Trichoderma Strains from Marine and Terrestrial Environments.

Nickel contamination is a serious environmental issue that requires immediate action. In this study, 23 strains of Trichoderma were isolated from terrestrial and marine environments and identified using a polyphasic approach of morphological characterization and ITS gene sequence analysis. The Trichoderma strains were tested for their tolerance and biosorption of nickel. Our results showed the growth of all Trichoderma strains on Trichoderma Selective Medium (TSM) with 50–1200-ppm nickel, indicating their tolerance of this heavy metal even at a relatively high concentration. Six Trichoderma strains (three isolated from terrestrial substrates and three from marine substates) had the highest radial growth on TSM with 50-ppm Ni. Among these fungal isolates, Trichoderma asperellum (S03) isolated from soil exhibited the best growth after 2 days of incubation. For the biosorption of nickel, the accumulation or uptake efficiency by the six selected Trichoderma was determined in Potato Dextrose Broth (PDB) supplemented with 50-ppm Ni using a Flame Atomic Absorption Spectrophotometer (AAS). The percent uptake efficiency of the three strains of T. asperellum (S03, S08, and LL14) was computed to be up to 66%, while Trichoderma virens (SG18 and SF22) and Trichoderma inhamatum (MW25) achieved up to 68% uptake efficiency. Observation of the Trichoderma strains with Scanning Electron Microscopy (SEM) before and after the absorption of nickel showed very minimal damage on the hyphal and conidial surface morphology, but changes in the colonial characteristics were observed. Our study highlighted the potential of terrestrial and marine strains of Trichoderma for the bioremediation of nickel pollution.

Emendation of the Coccoid Cyanobacterial Genus Gloeocapsopsis and Description of the New Species Gloeocapsopsis diffluens sp. nov. and Gloeocapsopsis dulcis sp. nov. Isolated From the Coastal Range of the Atacama Desert (Chile)

The taxonomy of coccoid cyanobacteria, such as Chroococcidiopsis, Pleurocapsa, Chroococcus, Gloeothece, Gloeocapsa, Gloeocapsopsis, and the related recent genera Sinocapsa and Aliterella, can easily be intermixed when solely compared on a morphological basis. There is still little support on the taxonomic position of some of the addressed genera, as genetic information is available only for a fraction of species that have been described solely on morphology. Modern polyphasic approaches that combine classic morphological investigations with DNA-based molecular analyses and the evaluation of ecological properties can disentangle these easily confusable unicellular genera. By using such an approach, we present here the formal description of two novel unicellular cyanobacterial species that inhabit the Coastal Range of the Atacama Desert, Gloeocapsopsis dulcis (first reported as Gloeocapsopsis AAB1) and Gloeocapsopsis diffluens. Both species could be clearly separated from previously reported species by 16S rRNA and 16S–23S ITS gene sequencing, the resulting secondary structures, p-distance analyses of the 16S–23S ITS, and morphology. For avoiding further confusions emendation of the genus Gloeocapsopsis as well as epitypification of the type species Gloeocapsopsis crepidinum based on the strain LEGE06123 were conducted.

Optimization of Conditions for Xylanase Production Using Aspergillus tubingensis Under Different Carbon Sources

Xylanases are hydrolytic enzymes with wide range of applications in food processing, bleaching of pulp in paper manufacturing industry, bio-conversion of biomass wastes to fermentable sugars and enhancing nutrient digestibility in animal feeds. The optimization of growth conditions and evaluation of an appropriate substrate as carbon source among cassava peels, corn cobs, wheat bran and rice husk on xylanase production by novel strain of Aspergillus tubingensis under Solid State Fermentation (SSF) was investigated. The fungal isolate was identified based on ribosomal RNA gene and ITS gene sequencing analysis as Aspergillus tubingensis. Results showed that Corn cobs had the highest xylanase production among the four substrates. Corn cobs recorded the highest value of xylanase production at pH of 6.0 (107.97 U/g), after incubation period of 72 hour (111.23 U/g), at temperature of 30oC (44.26 U/g) and at ratio 1:3 (45.68 U/g). The optimum growth conditions for xylanase production by Aspergillus tubingensis were: pH 6.0, incubation period of 72 hours, temperature of 30oC and substrate concentration of 1:3 (4g of substrate per 12ml of fermentation medium). Corn cobs showed the highest yield of xylanase activity (111.23±0.31 U/g), followed by Rice husk (101.91±0.72 U/g), Wheat bran (89.30±1.16 U/g) and Cassava peel (87.03±0.57 U/g). In conclusion, among the various agro residues that were used as carbon sources, Corn cobs had maximum xylanase activity. Various culture conditions were optimized by using one factor at a time method and the maximum xylanase production was obtained at pH of 6.0, incubation period of 72 hour, temperature of 30oC and substrate concentration of 1:3 under solid state fermentation. It is therefore suggested that some other notable environmental and fermentation factors that influence metabolism-mediated production yields of xylanase like aeration, agitation, carbon and nitrogen sources, metal ion requirement, inoculum size etc. should be optimized for maximum production of enzyme. Keywords: Solid State Fermentation, Cassava peels, Corn cobs, Wheat bran, Rice husk

Antibacterial Potential of Fungal Endophytes Isolated from Carica papaya

Background: Drug resistant bacteria related health problems are major concern globally and search for newer and most effective antibacterial agents is the urgent need to combat with these challenges. The study was undertaken with the aim of isolating and identifying endophytic fungi associated with Carica papaya and assessing their potential as antibacterial agents.Methods: Carica papaya plants were collected from different locations and endophytic fungi were isolated and characterized phenotypically and genotypically by microscopy, colonial characteristics and ITS gene sequencing, respectively. Antibacterial activity of endophyte was assessed against Escherichia coli, Klebsiella pneumonia, Salmonella Typhimurium, Bacillus cereus, Staphylococcus aureus and Streptococcus pyogenes, using the agar plate diffusion assay method with Ciprofloxacin as a positive control.Result: Thirty four (34) fungal endophytes of two genera Fusarium and Penicillium were recovered from 60 samples of Carica papaya and they showed the antibacterial activity against Staphylococcus aureus. Thus, the endophytic fungi have the potential to be used as an antibacterial agent.

Production of Mycophenolic Acid by a Newly Isolated Indigenous Penicillium glabrum.

Soil-occupant fungi produce a variety of mycotoxins as secondary metabolites, one of which is mycophenolic acid (MPA), an antibiotic and immunosuppressive agent. MPA is mainly produced by several species of Penicillium, especially Penicillium brevicompactum. Here, we present the first report of MPA production by a local strain belonging to Penicillium glabrum species. We screened ascomycete cultures isolated from moldy food and fruits, as well as soils, collected from different parts of Iran. MPA production of one hundred and forty Penicillium isolates was analyzed using HPLC. Three MPA producer isolates were identified, among which the most producer was subjected to further characterization, based on morphological and microscopic analysis, as well as molecular approach (ITS, rDNA and beta-tubulin gene sequences). The results revealed that the best MPA producer belongs to P. glabrum IBRC-M 30518, and can produce 1079 mg/L MPA in Czapek-Dox medium.

<p><strong>Polyphasic taxonomy of Indian Trichoderma species</strong></p>

Trichoderma speies are known for their diverse applications as potent bio-control organisms. 34 isolates of Trichoderma were obtained from various geographical locations in India and were subjected to phenotypic evaluation. Species identification if based on only morphology, may lead to sometimes erroneous, uncertain or remains unclear due to overlapping characters. Therefore, molecular characterization was also performed based on ITS region sequence analysis for reliable identification using both morphological and molecular characters. The isolates were identified into 20 different species viz., T. aggressivum, T. asperellum, T. atroviride, T. brevicompactum, T. citrinoviride, T. crassum, T. erinaceum, T. ghanense, T. hamatum, T. harzianum, T. koningiopsis, T. longibrachiatum, T. longipile (Syn. Hypocrea longipilosa), T. minutisporum, T. pubscenes, T.reesei, T. saturnisporum, T. spirale, T. tomentosum and T. virens based on the integrated approach of both morphological and molecular characterisation. ß-tub gene sequence analysis was also performed to find out the phylogenetic relationship of these species. The ß-tub gene sequences differentiated all the 20 different species of Trichoderma better than the ITS region sequences. The morphological characters viz., colony, conidiophores, phialides, conidia and chlamydospores of all the twenty species of Trichoderma were described along with photomicrographs. The ITS and β-tub gene sequences of all the 20 species studied have been deposited in the Genbank.

Diaporthe taoicola and D. siamensis, Two New Records on Citrus sinensis in China

Two Diaporthe species isolated from fruit of Citrus sinensis in China were characterized based on morphology and multilocus phylogeny of ITS, tef1, and tub2 gene sequences. The phylogeny indicated that the two species match Diaporthe taoicola and D. siamensis. A critical examination of phenotypic characteristics confirmed the phylogenetic results. Diaporthe taoicola was morphologically characterized by producing Alpha conidia with tapering toward both ends. Meanwhile, D. siamensis produced cylindrical or ellipsoidal Alpha conidia with two oil drops. Pathogenicity tests revealed that both species were pathogenic to fruit of C. sinensis. To our knowledge, the two species were firstly reported on Citrus sinensis in China.

Watanabeales ord. nov. and twelve novel species of Trebouxiophyceae (Chlorophyta).

The Trebouxiophyceae include Chlorellales, Prasiolales, Trebouxiales, Microthamniales, and numerous members for which taxonomic locations have not been determined. The Watanabea clade is a group in the Trebouxiophyceae of Chlorella-like taxa, which are predominantly solitary and terrestrial. With the increase in descriptions of new species within the Watanabea clade in recent years, it is necessary to define the boundary and taxonomic status of the clade. In this study, nineteen strains of terrestrial algae collected from China were investigated based on molecular and morphological data, involving phylogenetic analyses, light and electron microscopy, and ITS-2 secondary structures. These 19 strains were described as five known species and 12 novel ones. Based on the consistent topological structures of the phylogenetic analyses for three taxonomic levels (the core Chorophyta, Trebouxiophyceae based on 18S sequences, Watanabeales and Symbiochloris based on 18S, 5.8S, ITS2 and rbcL gene sequences) and common morphological characteristics, especially the key phenotypic feature of unequally sized autospores, the Watanabea clade was defined as a new order, Watanabeales ord. nov., with one family, Watanabeaceae fam. nov. The boundary of Watanabeales was delineated and included the genera Chloroidium, Calidiella, Jaagichlorella, Kalinella, Massjukichlorella, Mysteriochloris, Polulichloris, Phyllosiphon, Watanabea, and Viridiella. Symbiochloris did not belong to Watanabeales according to the phylogenetic analyses and the reproduction by equal-sized autospores, aplanospores, or zoospores. The Watanabeales were separated into four groups during the phylogenetic analyses, but no regular differences in habitats or morphology could be used as the morphological basis to split Watanabeales into the four groups.

Uncovering the hidden bacterial ghost communities of yeast and experimental evidences demonstrates yeast as thriving hub for bacteria

Our major concern was to address “yeast endobacteria” which was based on a few reports in the recent past where bacteria may find yeast as a niche for survival. In this study, we report the microbiota of twenty-nine axenic yeast cultures recovered from different habitats based on their 16S rRNA gene-amplicon metagenomes. Yeasts were identified based on D1/D2 or ITS gene sequences. Bacterial diversity was widespread, varied and rich among all yeasts except for four strains. Taxa belonging to the phylum Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes and the genera; Streptococcus, Propionibacterium were common to all the yeasts. Candida tropicalis was used as a model organism to confirm bacteria through fluorescence in situ hybridization (FISH), isolating and re-introducing the isolated bacteria into the yeast. FISH analysis confirmed the endobacteria of C. tropicalis and we have successfully isolated four bacteria only after lysis and disruption of yeast cells. These bacteria were identified as species of Pseudomonas, Chryseobacterium, Lysinibacillus and Propionibacterium. Guestimates indicate 95% of bacterial species of C. tropicalis are yet-to-be-cultivated. We have successfully reintroduced mCherry tagged Pseudomonas into C. tropicalis. Also, auto-fluorescent Prochlorococcus and Rhodopseudomonas could be introduced into C. tropicalis while mCherry tagged E. coli or Salmonella could not be introduced. FISH analysis confirmed the presence of both native and infected bacterial cells present in C. tropicalis. Our findings unveil the insights into the ghost microbiota associated with yeast, which otherwise are considered to be axenic cultures. Their inherent occurrence, together with co-cultivation experiments under laboratory conditions suggests that yeasts are a thriving hub for bacterial communities.