Comparison of DNA and RNA, and Cultivation Approaches for the Recovery of Terrestrial and Aquatic Fungi from Environmental Samples

Estimates of fungal biodiversity from environmental samples are all subject to bias. Major issues are that the commonly adopted cultivation-based approaches are suitable for taxa which grow readily under laboratory conditions, while the DNA-based approaches provide more reliable estimates, but do not indicate whether taxa are metabolically active. In this study, we have evaluated these approaches to estimate the fungal diversity in soil and freshwater samples from a subtropical forest, and compared these to RNA-based culture-independent approach intended to indicate the metabolically active fungal assemblage. In both soil and freshwater samples, the dominant taxon recovered by all three approaches was the same (Anguillospora furtiva). This taxon was cultivable from all samples and comprised 85–86 % DNA libraries and 90–91 % RNA libraries. The remaining taxa were phylogenetically diverse and spanned the Ascomycota, Basidiomycota, and Fungi incertae sedis. Their recovery was not consistent among the three approaches used and suggests that less abundant members of the assemblage may be subjected to greater bias when diversity estimates employ a single approach. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00284-012-0256-7) contains supplementary material, which is available to authorized users

Optimal conditions of mycelial growth of three wild edible mushrooms from northern Thailand

In this study, three wild mushrooms namely Lentinus connatus, L. roseus, and Pleurotusgiganteus were selected to study if they could be domesticated. Initially, the fruiting bodies of the three mushrooms were collected from forests in northern Thailand and morphologically characterized. In this paper we report the optimal in vitro culture conditions of three wild mushrooms. Among seven culture media tested for the optimal mycelial growth of three wild mushrooms, black bean agar, red bean and soy bean agar were the best for the mycelial growth of L. connatus, L. roseus and Pleurotusgiganteus, respectively. The mushroom mycelia were able to grow at temperatures ranging from 20-30 °C, with optimal growth temperatures of 30 °C and 25 °C for Lentinus and Pleurotus species, respectively. The optimum pH range observed for mycelial growth was 5.0 - 7.0

LMC X-1: A New Spectral Analysis of the O-star in the binary and surrounding nebula

We provide new observations of the LMC X-1 O star and its extended nebula structure using spectroscopic data from VLT/UVES as well as H$\alpha$ imaging from the Wide Field Imager on the Max Planck Gesellschaft / European Southern Observatory 2.2m telescope and ATCA imaging of the 2.1 GHz radio continuum. This nebula is one of the few known to be energized by an X-ray binary. We use a new spectrum extraction technique that is superior to other methods to obtain both radial velocities and fluxes. This provides an updated spatial velocity of $\simeq 21.0~\pm~4.8$ km s$^{-1}$ for the O star. The slit encompasses both the photo-ionized and shock-ionized regions of the nebula. The imaging shows a clear arc-like structure reminiscent of a wind bow shock in between the ionization cone and shock-ionized nebula. The observed structure can be fit well by the parabolic shape of a wind bow shock. If an interpretation of a wind bow shock system is valid, we investigate the N159-O1 star cluster as a potential parent of the system, suggesting a progenitor mass of $\sim 60$ M$_{\odot}$ for the black hole. We further note that the radio emission could be non-thermal emission from the wind bow shock, or synchrotron emission associated with the jet inflated nebula. For both wind and jet-powered origins, this would represent one of the first radio detections of such a structure.Comment: 7 Figures, 4 Table

Extravehicular Activity Micrometeoroid and Orbital Debris Risk Assessment Methodology

A well-known hazard associated with exposure to the space environment is the risk of vehicle failure due to an impact from a micrometeoroid and orbital debris (MMOD) particle. Among the vehicles of importance to NASA is the extravehicular mobility unit (EMU) spacesuit used while performing a US extravehicular activity (EVA). An EMU impact is of great concern as a large leak could prevent an astronaut from safely reaching the airlock in time resulting in a loss of life. For this reason, a risk assessment is provided to the EVA office at the Johnson Space Center (JSC) prior to certification of readiness for each US EVA

A multi-gene phylogeny of Lactifluus (Basidiomycota, Russulales) translated into a new infrageneric classification of the genus

Infrageneric relations of the genetically diverse milkcap genus Lactifluus (Russulales, Basidiomycota) are poorly known. Currently used classification systems still largely reflect the traditional, mainly morphological, characters used for infrageneric delimitations of milkcaps. Increased sampling, combined with small-scale molecular studies, show that this genus is underexplored and in need of revision. For this study, we assembled an extensive dataset of the genus Lactifluus, comprising 80 % of all known species and 30 % of the type collections. To unravel the infrageneric relationships within this genus, we combined a multi-gene molecular phylogeny, based on nuclear ITS, LSU, RPB2 and RPB1, with a morphological study, focussing on five important characteristics (fruit body type, presence of a secondary velum, colour reaction of the latex/context, pileipellis type and presence of true cystidia). Lactifluus comprises four supported subgenera, each containing several supported clades. With extensive sampling, ten new clades and at least 17 new species were discovered, which highlight the high diversity in this genus. The traditional infrageneric classification is only partly maintained and nomenclatural changes are proposed. Our morphological study shows that the five featured characteristics are important at different evolutionary levels, but further characteristics need to be studied to find morphological support for each clade. This study paves the way for a more detailed investigation of biogeographical history and character evolution within Lactifluus

pncA and bptA Are Not Sufficient To Complement Ixodes scapularis Colonization and Persistence by Borrelia burgdorferi in a Linear Plasmid lp25-Deficient Background

The complex segmented genome of Borrelia burgdorferi is comprised of a linear chromosome along with numerous linear and circular plasmids essential for tick and/or mammalian infectivity. The pathogenic necessity for specific borrelial plasmids has been identified; most notably, infections of the tick vector and mammalian host both require linear plasmid 25 (lp25). Genes carried on lp25, specifically bptA and pncA, are postulated to play a role for B. burgdorferi to infect and persist in Ixodes ticks. In this study, we complemented an lp25-deficient borrelial strain with pncA alone or pncA accompanied by bptA to evaluate the ability of the complemented strains to restore larval colonization and persistence through transstadial transmission relative to that of wild-type B. burgdorferi. The acquisition of the complemented strains by tick larvae from infected mice and/or the survival of these strains was significantly decreased when assayed by cultivation and quantitative PCR (qPCR). Only 10% of the pncA-complemented strain organisms were found by culture to survive 17 days following larval feeding, while 45% of the pncA- and bptA-complemented strain organisms survived, with similar results by PCR. However, neither of the complemented B. burgdorferi strains was capable of persisting through the molt to the nymphal stage as analyzed by culture. qPCR analyses of unfed nymphs detected B. burgdorferi genomes in several nymphs at low copy numbers, likely indicating the presence of DNA from dead or dying cells. Overall, the data indicate that pncA and bptA cannot independently support infection, suggesting that lp25 carries additional gene(s) or regulatory elements critical for B. burgdorferi survival and pathogenesis in the Ixodes vector

Ribosomal and Protein Gene Phylogeny Reveals Novel Saprobic Fungal Species FromJuglans regiaandUrtica dioica

During an ongoing investigation of Ascomycetes from plant substrates, three saprobic species were found from plant substrates. Two new species,Leptosphaeria regiaeandNeomicrosphaeropsis juglandiswere isolated from dead branches ofJuglans regiafrom Turkey. Another species is introduced herein asSubplenodomus urticaesp. nov within the family Leptosphaeriaceae found onUrtica dioicain Italy. Multigene phylogenies based on combined LSU, ITS, SSU, and beta-tubulin DNA sequence data generated from maximum likelihood and MrBayes analyses indicate thatLeptosphaeria regiaeis related toL. slovacicaand forms an independent lineage within the genusLeptosphaeria.Subplenodomus urticaeis basal toS. iridicolaand its establishment as a new species is strongly supported.Neomicrosphaeropsis juglandisforms a moderately supported lineage in betweenN. italicaandN. elaeagniin the Didymellaceae. Full morphological details are provided herein and phylogenetic relationships of the three new species are also discussed

Incorporating molecular data in fungal systematics: a guide for aspiring researchers

The last twenty years have witnessed molecular data emerge as a primary research instrument in most branches of mycology. Fungal systematics, taxonomy, and ecology have all seen tremendous progress and have undergone rapid, far-reaching changes as disciplines in the wake of continual improvement in DNA sequencing technology. A taxonomic study that draws from molecular data involves a long series of steps, ranging from taxon sampling through the various laboratory procedures and data analysis to the publication process. All steps are important and influence the results and the way they are perceived by the scientific community. The present paper provides a reflective overview of all major steps in such a project with the purpose to assist research students about to begin their first study using DNA-based methods. We also take the opportunity to discuss the role of taxonomy in biology and the life sciences in general in the light of molecular data. While the best way to learn molecular methods is to work side by side with someone experienced, we hope that the present paper will serve to lower the learning threshold for the reader.Comment: Submitted to Current Research in Environmental and Applied Mycology - comments most welcom

First Report of Phaeoacremonium minimum Associated with Grapevine Trunk Diseases in China

Grapevine trunk diseases (GTDs) are a complex of diseases that strongly affect grape productivity, wine quality, and vineyard longevity worldwide. GTDs occur in diverse regions, and in some areas disease agents are present in up to 100% of vines (Pintos et al. 2018). Recently, species belonging to Botryosphaeria and Diaporthe were identified as pathogens associated with GTDs in China (Dissanayake et al. 2015; Yan et al. 2013). During 2017 to 2018, interveinal leaf necrosis and wood white decay surrounded by brown necrosis in longitudinal section and black spots in cross section were observed in several grapevines (Cabernet) in Huailai County, Hebei Province, China. Disease incidence was up to 2 to 3% of the vineyard. Diseased samples (whole trunk and roots of 19-year-old vines) were collected and taken to the laboratory. Woody samples were cut into small chips, surface sterilized in 1.5% NaOCl for 3 min followed by 70% ethanol for 30 s, and rinsed three times with sterile distilled water (SDW). Once the samples were dried, they were placed onto PDA plates amended with ampicillin (0.1 g/liter). Plates were incubated at 25°C under dark conditions. After 14 days of incubation, hyphal tips of fungi growing from wood pieces were transferred onto new PDA plates and incubated until they produced conidia. One type of colony was consistently isolated from the discolored tissue, with honey brown mycelium, and producing a yellow pigment on PDA. Conidia were ellipsoid to allantoid, 3.8 to 6.2 μm long, and 1.6 to 3.2 μm wide (n = 50). Morphologically these isolates resembled species belonging to Phaeoacremonium (Mostert et al. 2006). For species confirmation, genomic DNA of three representative isolates (JZB3190001, JZB3190003, and JZB3190005) was extracted. PCR amplification was performed using two phylogenetic markers (actin and β-tubulin) amplified with primers ACT-513F/ACT-783R (Carbone and Kohn 1999) and T1/Bt2b (Glass and Donaldson 1995). The sequences obtained were deposited in GenBank under the accession numbers MK994188 to MK994193. Phylogenetic analysis was conducted using maximum likelihood in RAxML, which was accomplished using RAxML-HPC2 on XSEDE in the CIPRES Science Gateway platform (http://www.phylo.org/). In the phylogenetic tree, the isolates from the present study clustered together with Phaeoacremonium minimum (CBS 246.91), with 100 bootstrap values. Based on morphological characters and phylogenetic results, the species isolated in this study was identified as P. minimum (Tul. & C. Tul.) D. Gramaje, L. Mostert & Crous (Gramaje et al. 2015). The pathogenicity test was conducted on healthy, 2-month-old, rooted 'Furcal' grapevines that were grown in an inoculation chamber. Roots were washed using flow water and rinsed with SDW. The ends of SDW-treated roots (roughly 1 cm) were cut with sterilized scissors, and the roots and trunk base of Furcal vines were dipped in a 10⁶/ml P. minimum spore suspension for 30 min. Inoculated plants were immediately planted in individual pots, and 10 ml of spore suspension was added to the soil per pot. SDW was used as a control. Two P. minimum isolates (JZB3190001 and JZB3190003) were used to do the pathogenicity test, and 10 plants were inoculated with each isolate and the control water. Temperature of the inoculation chamber was controlled between 24 and 25°C, and humidity was maintained at 60%. After 114 days of inoculation, the inoculated plants developed black necrosis at the base of wood but did not show leaf necrosis, whereas the control plants showed no symptoms both in wood and leaves. Koch's postulates were confirmed by reisolating and identification based on cultural and morphological characters of the inoculated isolates. To our knowledge, this is the first report of P. minimum associated with GTDs in China. The results of this study will enhance the capability of controlling GTDs in China by correct identification of the causal organism

Pleosporales

published_or_final_versionSpringer Open Choice, 25 May 201