Biological and chemical diversity of cytotoxin-producing symbiotic marine fungi in intertidal zone of Dalian

Abstract. Radiastuti N, Bahalwan HA, Susilowati DN. 2019. Phylogenetic study of endophytic fungi associated with Centella asiatica from Bengkulu and Malaysian accessions based on the ITS rDNA sequence. Biodiversitas 20: 1248-1258. Centella asiatica is one of the medicinal plants which is known to be symbiotic with various endophytic fungi. The purpose of this research was to determine diversity of culturable fungal endophyte from C. asiatica. Identification was conducted using molecular phylogenetic analysis based on the ITS rDNA sequence. The result showed that obtained 145 isolates endophytic fungi (from stolons, leaves, roots, petioles) were grouped into 18 morphotaxa (Bengkulu) and 23 morphotaxa (Malaysian). The fungal endophytic were identified as Aspergillus austroafricanus, Aspergillus oryzae, Acrocalymma vagum, Ceratobasidium cornigerum, Ceratobasidium sp., Chaetomium globosum, Colletotrichum karstii, C. gigasporium, C. tabaci, Colletotrichum siamense, Eutypella sp., Fusarium solani, F. oxysporum, F. falciforme, F. keratoplasticum, F. striatum, Fusarium sp., Penicillium capsulatum, Phoma multirostrata, Perenniporia tephropora, Perenniporia sp., Phanerochaete chrysosporium, Phanerochaete stereoides, Phyllosticta capitalensis, Phomopsis asparagi, Peroneutypa scoparia, Phialemoniopsis sp., Mycochaetophora gentinae, Talaromyces sp., Earliella scabrosa, and Trichaptum sp. based on molecular phylogenetic analysis. Fusarium (Nectriaceae) were the most found of fungal endophytes in both C. asiatica Bengkulu and Malaysian accession. The majority strain are including Ascomycetes and Basidiomycetes. The identification of endophytic fungi from medicinal plants is needed as a preliminary study to determine the potential of endophytic fungi producing bioactive compounds.

Arguably more mycophiles hunt true morels (Morchella) during their brief fruiting season each spring in the northern hemisphere than any other wild edible fungus. Concerns about overharvesting by individual collectors and commercial enterprises make it essential that science-based management practices and conservation policies are developed to ensure the sustainability of commercial harvests and to protect and preserve morel species diversity. Therefore, the primary objectives of the present study were to: (i) investigate the utility of the ITS rDNA locus for identifying Morchella species, using phylogenetic species previously inferred from multilocus DNA sequence data as a reference; and (ii) clarify insufficiently identified sequences and determine whether the named sequences in GenBank were identified correctly. To this end, we generated 553 Morchella ITS rDNA sequences and downloaded 312 additional ones generated by other researchers from GenBank using emerencia and analyzed them phylogenetically. Three major findings emerged: (i) ITS rDNA sequences were useful in identifying 48/62 (77.4%) of the known phylospecies; however, they failed to identify 12 of the 22 species within the species-rich Elata Subclade and two closely related species in the Esculenta Clade; (ii) at least 66% of the named Morchella sequences in GenBank are misidentified; and (iii) ITS rDNA sequences of up to six putatively novel Morchella species were represented in GenBank. Recognizing the need for a dedicated Web-accessible reference database to facilitate the rapid identification of known and novel species, we constructed Morchella MLST (http://www.cbs.knaw.nl/morchella/), which can be queried with ITS rDNA sequences and those of the four other genes used in our prior multilocus molecular systematic studies of this charismatic genus.

Endolichenic fungi are microbes that inhabit healthy inner lichen tissues without any disease symptoms. They have been reported to produce new and interesting bioactive metabolites. In the present study, an endolichenic fungus frequently isolated from surface-sterilized lichen thallus of Parmelia caperata has been described. The fungus was identified as Aspergillus tubingensis based on morphological traits and ITS rDNA sequence. Crude metabolites extracted from the culture broth exhibited considerable antimicrobial activity against a panel of clinically significant human pathogens. The fungus showed optimum antimicrobial activity in PDB medium in day 7 of incubation period. PDB medium amended with 1% NaCl and at alkaline pH was found to be optimal for antimicrobial metabolites production. Enhanced activity was observed when the fungus was exposed briefly to a heat shock of 60°C during incubation. The metabolites showed optimum λ-max at 214nm with an absorbance value of 1.589. Molecular characterization of the isolate was carried out by ITS phylogeny and ITS2 secondary structure analyses. The phylogenetic trees based on both ITS rDNA and ITS2 sequences showed the isolate within the clade A. tubingensis. Considering the ubiquity and ambiguity in identifying Aspergillus species of different lifestyles, a method to differentiate pathogenic and endophytic Aspergillus at species level was developed using ITS2 secondary structure analysis. The results showed common folding pattern in the secondary structures with a helix and a 5' dangling end found to be highly conserved. Certain features in the secondary structure like multi-bulges and a symmetric interior loop were observed to be unique which distinguish our isolate from other A. tubingensis.

Symbiosis plays a fundamental role in nature. Lichens are among the best known, globally distributed symbiotic systems whose ecology is shaped by the requirements of all symbionts forming the holobiont. The widespread lichen-forming fungal genus Stereocaulon provides a suitable model to study the ecology of microscopic green algal symbionts (i.e., phycobionts) within the lichen symbiosis. We analysed 282 Stereocaulon specimens, collected in diverse habitats worldwide, using the algal ITS rDNA and actin gene sequences and fungal ITS rDNA sequences. Phylogenetic analyses revealed a great diversity among the predominant phycobionts. The algal genus Asterochloris (Trebouxiophyceae) was recovered in most sampled thalli, but two additional genera, Vulcanochloris and Chloroidium, were also found. We used variation-partitioning analyses to investigate the effects of climatic conditions, substrate/habitat characteristic, spatial distribution and mycobionts on phycobiont distribution. Based on an analogy, we examined the effects of climate, substrate/habitat, spatial distribution and phycobionts on mycobiont distribution. According to our analyses, the distribution of phycobionts is primarily driven by mycobionts and vice versa. Specificity and selectivity of both partners, as well as their ecological requirements and the width of their niches, vary significantly among the species-level lineages. We demonstrated that species-level lineages, which accept more symbiotic partners, have wider climatic niches, overlapping with the niches of their partners. Furthermore, the survival of lichens on substrates with high concentrations of heavy metals appears to be supported by their association with toxicity-tolerant phycobionts. In general, low specificity towards phycobionts allows the host to associate with ecologically diversified algae, thereby broadening its ecological amplitude.

This article has been retracted by the publisher.

Research on renewable biotechnology for renewable biofuel applications has reached new heights. This is highlighted by extensive biomining for novel enzymes to reduce the production costs from animal and insect gut microbiomes. This study explored the diversity and composition of hemicellulolytic fungi in the gut microbiota from dung beetles of the family Scarabaeidae (Pachylomerus femoralis, Anachalcos convexus and Euoniticellus intermedius). Two hundred and twenty-two filamentous fungi were isolated, purified and identified using rDNA sequencing of the ITS and D1/D2 regions. The fungal isolates were assigned to 12 genera and 25 species. Fungi associated with the genus Aspergillus was in abundance, with Hypocrea lixii predominantly isolated. Isolates that produced more than 3 U/mL of xylanase activity were evaluated further. The highest xylanase activity was of 23.6 and 23.5 U/mL for L1XYL9 (E. intermedius larvae) and Hypocrea lixii AB2A3 (A. convexus), respectively. Phylogeny of the fungal strains with xylanolytic activity was analysed using ITS rDNA sequences and revealed close genetic relatedness between isolates from the different dung beetle species. Fungal genera commonly found in the gut of both adult beetles and larvae included Aspergillus, Hypocrea, Talaromyces and Penicillium. The results obtained in this study suggest that the gut of Scarabaeidae dung beetles in South Africa is a rich source of xylanolytic fungi.

Cryptic speciation in Protoceratium reticulatum (Dinophyceae): Evidence from morphological, molecular and ecophysiological data

Identification of photobionts from the lichen family Physciaceae using algal-specific ITS rDNA sequencing

Research on the molecular systematics of Cortinarius, a species-rich mushroom genus with nearly global distribution, is just beginning. The present study explores infrageneric relationships using rDNA ITS and LSU sequence data. One large dataset of 132 rDNA ITS sequences and one combined da-taset with 54 rDNA ITS and LSU sequences were generated. Hebeloma was used as outgroup. Bayesian analyses and maximum-likelihood (ML) analyses were carried out. Bayesian phylogenetic inference performed equally well or better than ML, especially in large datasets. The phylogenetic analysis of the combined dataset with species representing all currently recognized subgenera recovered seven well-supported clades (Bayesian posterior probabilities BPP > 90%). These major clades are: /Myxacium s.l., /subg. Cortinarius, the /phlegmacioid clade (including the subclades /Phlegmacium and /Delibuti), the /calochroid clade (/Calochroi, /Ochroleuci and /Allutus), the /telamonioid clade (/Telamonia, /Orellani, /Anomali), /Dermocybe s.l. and /Myxotelamonia. Our results show that Cortinarius consists of many lineages, but the relationships among these clades could not be elucidated. On one hand, the low divergence in rDNA sequences can be held responsible for this; on the other hand, taxon sampling is problematic in Cortinarius phylogeny. Because of the incredibly high diversity (~2000 Cortinarius species), our sampling included <5% of the known species. By choosing type species of subgenera and sections, our sampling is strongly biased toward Northern Hemisphere taxa. More extensive taxon sampling, especially of species from the Southern Hemisphere, is essential to resolve the phylogeny of this important genus of ectomycorrhizal fungi.

Research on the molecular systematics of Cortinarius, a species-rich mushroom genus with nearly global distribution, is just beginning. The present study explores infrageneric relationships using rDNA ITS and LSU sequence data. One large dataset of 132 rDNA ITS sequences and one combined da-taset with 54 rDNA ITS and LSU sequences were generated. Hebeloma was used as outgroup. Bayesian analyses and maximum-likelihood (ML) analyses were carried out. Bayesian phylogenetic inference performed equally well or better than ML, especially in large datasets. The phylogenetic analysis of the combined dataset with species representing all currently recognized subgenera recovered seven well-supported clades (Bayesian posterior probabilities BPP > 90%). These major clades are: /Myxacium s.l., /subg. Cortinarius, the /phlegmacioid clade (including the subclades /Phlegmacium and /Delibuti), the /calochroid clade (/Calochroi, /Ochroleuci and /Allutus), the /telamonioid clade (/Telamonia, /Orellani, /Anomali), /Dermocybe s.l. and /Myxotelamonia. Our results show that Cortinarius consists of many lineages, but the relationships among these clades could not be elucidated. On one hand, the low divergence in rDNA sequences can be held responsible for this; on the other hand, taxon sampling is problematic in Cortinarius phylogeny. Because of the incredibly high diversity (~2000 Cortinarius species), our sampling included <5% of the known species. By choosing type species of subgenera and sections, our sampling is strongly biased toward Northern Hemisphere taxa. More extensive taxon sampling, especially of species from the Southern Hemisphere, is essential to resolve the phylogeny of this important genus of ectomycorrhizal fungi.

We used a combination of molecular-phylogenetic inference of 82 ITS rDNA sequences and anatomical approach to describe three new west African thelephoroid species, namely Tomentella afrostuposa, T. guineensis and T. guinkoi. Anatomically, T. afrostuposa is reminiscent of T. stuposa with globose to broadly ellipsoid large basidiospores of 8–14 μm, long aculei of up to 3 μm and prominent apiculi of 2 μm width. Molecular-phylogenetically, it falls within the T. stuposa complex. However, T. afrostuposa deviates by at least 7.80–10.74% from T. stuposa in regard with the ITS rDNA sequences. Tomentella guineensis is characterised by long (up to 85 μm) utriform basidia, the presence of reniform basidiospores in lateral view (up to 9 μm) with aculei not exceeding 1 μm and a strong cyanescent reaction of the subhymenial hyphae and basidia in 2.5% KOH. It forms a sister species of the newly described species Tomentella maroana; however, deviating from the last species by at least 9.75–10.04%. The very short, inflated (up to 14 μm) and thick-walled septate (septa up to 1.5 μm) subhymenial hyphae combined with ellipsoid basidiospores (up to 8 μm) and short aculei not exceeding 0.5 μm characterise Tomentella guinkoi. Anatomically, T. guinkoi recalls T. ellisii. Genetic distance between both species ranges from 12.67 to 13.73% according to ITS rDNA sequences analyses. Tomentella guinkoi forms a sister species of the group composed of T. ellisii, T. hjortstamiana and T. pisoniae. Detailed anatomical comparisons between the newly described species and their close relatives are given.

Sclerotinia stem rot caused by necrotrophic plant pathogenic fungus Sclerotinia sclerotiorum (Lib) de Bary is a highly pervasive disease affecting a wide range of agricultural and horticultural crops. In this study, fungal endophytes recovered from inflorescence tissues of Aloe vera L. were examined for their antagonist activity against S. sclerotiorum and the presence of plant growth-promoting traits. Fungal endophytes were characterized by microscopic and molecular (ITS rDNA sequencing) methods. Chaetomium globosum was the predominant endophytic species followed by Trichoderma harzanium in this study. Trichoderma harzanium tested positive for the presence of plant growth-promoting traits, i.e. siderophore, indole acetic acid, hydrogen cyanide production and phosphate solubilization. Chaetomium globosum strain 2 and T. harzanium survived stressful conditions (high temperature, cold stress and UV-B exposure). In antagonism assay, T. harzanium exhibited the highest growth inhibition per cent of 81.01 ± 0.63 against S. sclerotiorum and also restricted sclerotia formation. The hexane extract of T. harzanium (TH hex) obtained from solid fermentation depicted best antifungal activity against phytopathogen having an IC50 value of 3.987 mg/ml which is moderately comparable with positive control carbendazim (50%, WP). The GC–MS analysis of TH hex detected abundant presence (39.4%) of hexadecanoic acid, 2, 3-bis [(trimethylsilyl) oxy] propyl ester and other unsaturated fatty acids (palmitic and linoleic acid) having antifungal activity. The study demonstrates antifungal activity and plant growth-promoting potential of fungal endophytes residing in the inflorescence of A. vera.

Impact of Domestication on the Endophytic Fungal Diversity Associated With Wild Zingiberaceae at Mount Halimun Salak National Park

BackgroundArbuscular mycorrhizal fungi (AMF) can form obligate symbioses with the vast majority of land plants, and AMF distribution patterns have received increasing attention from researchers. At the local scale, the distribution of AMF is well documented. Studies at large scales, however, are limited because intensive sampling is difficult. Here, we used ITS rDNA sequence metadata obtained from public databases to study the distribution of AMF at continental and global scales. We also used these sequence metadata to investigate whether host plant is the main factor that affects the distribution of AMF at large scales.ResultsWe defined 305 ITS virtual taxa (ITS-VTs) among all sequences of the Glomeromycota by using a comprehensive maximum likelihood phylogenetic analysis. Each host taxonomic order averaged about 53% specific ITS-VTs, and approximately 60% of the ITS-VTs were host specific. Those ITS-VTs with wide host range showed wide geographic distribution. Most ITS-VTs occurred in only one type of host functional group. The distributions of most ITS-VTs were limited across ecosystem, across continent, across biogeographical realm, and across climatic zone. Non-metric multidimensional scaling analysis (NMDS) showed that AMF community composition differed among functional groups of hosts, and among ecosystem, continent, biogeographical realm, and climatic zone. The Mantel test showed that AMF community composition was significantly correlated with plant community composition among ecosystem, among continent, among biogeographical realm, and among climatic zone. The structural equation modeling (SEM) showed that the effects of ecosystem, continent, biogeographical realm, and climatic zone were mainly indirect on AMF distribution, but plant had strongly direct effects on AMF.ConclusionThe distribution of AMF as indicated by ITS rDNA sequences showed a pattern of high endemism at large scales. This pattern indicates high specificity of AMF for host at different scales (plant taxonomic order and functional group) and high selectivity from host plants for AMF. The effects of ecosystemic, biogeographical, continental and climatic factors on AMF distribution might be mediated by host plants.

Background: Plastic has become one of the most significant environmental threats, especially in mangrove areas due to their non-biodegradable nature. This indicates that there is a need to find alternative degradation methods for these materials, such as the use of mold. Therefore, this study aims to isolate potential plastic degradation mold from the Wonorejo mangrove sediments.Methods: The isolates were identified based on their morphological characteristic and ITS rDNA sequence. Plastic degradation ability of the samples was evaluated using parameters of plastic dry weight loss, FTIR pattern, SEM, and AFM image.Results: A total of eight sediment mold were identified, including Perenniporia spp., Trametes polyzona, Aspergillus terreus, Porostereum spadiceum, Leptosphaerulina chartarum, Aspergillus fumigatus, and Hypocreales sp. Among these molds, the best activity was exhibited by Aspergillus terreus, which degraded 12.5% LDPE and 4.9% white plastic after 30 days, followed by Perenniporia sp. with 10.6% LDPE and 3.4% white plastic degradation. Furthermore, the SEM and AFM images showed that the surface of plastic was damaged after incubation. There were also several attenuations of particular peaks, which indicated the occurrence of chemical changes along plastic chains belonging to the C-H alkane group at wavenumber 2914.94, 2847.38, and 1471 cm-1. Enzyme produced by mold in this study were measured qualitatively. Perenniporia sp. produced laccase, manganese peroxidase, alkane hydroxylase, and lipase. Trametes polyzona also secreted similar enzyme, except lipase, while Porostereum spadiceum was negative for alkane hydroxylase. Aspergillus terreus isolates were positive for lipase and alkane hydroxylase, but Aspergillus fumigatus only produced lipase.Conclusion: In this study, 8 mold with plastic biodegradation potential by producing various enzyme were identified.Keywords: Biodegradation, Enzyme, Mangrove; Mold; Plastic; Responsible consumption &amp; production